Parallel proximity ligation event analysis

ABSTRACT

The present invention describes compositions and methods showing that the spatial proximity of intracellular components may be related to their ability to cooperate in intracellular biochemical reactions. In some embodiments, the present invention contemplates a variety of nucleic acid barcoded binding partners capable of determining the spatial proximity of intracellular components as determined by ligation of their respective nucleotide barcodes. As such, an intracellular component contact map may be constructed to fingerprint specific physiological and/or pharmacological intracellular conditions.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number5-U54-HG003067-06 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

FIELD OF INVENTION

The present invention is related to the field of intracellularbiochemistry. For example, the spatial proximity of intracellularcomponents may be related to their ability to cooperate in intracellularbiochemical reactions. In some embodiments, the present inventioncontemplates a variety of nucleic acid barcoded binding partners capableof determining the spatial proximity of intracellular components asdetermined by ligation of their respective nucleotide barcodes. As such,an intracellular component contact map may be constructed to fingerprintspecific physiological and/or pharmacological intracellular conditions.

BACKGROUND

It is often of great interest to determine whether two cellularcomponents are in close proximity to one another. For instance, thereare several existing methods for determining proteins with the tendencyto form complexes, such as Yeast 2-hybrid and mass spectroscopy. Thesemethods require separate experiments for each protein of interest andcannot be used to probe other cellular components such as nucleic acids(DNA and RNA) and small molecules. In addition, they do not takeadvantage of the plummeting cost of sequencing.

Further, current methods to provide intracellular localization ofindividual proteins and/or enzymes only provides partial information.These studies have no relevance as to whether these proteins areactively participating in an intracellular biochemical function, ormerely being transported from one intracellular region to another.Further, these methods do not provide information regarding hundreds ifnot thousands of biochemical functions that are occurringsimultaneously.

Clearly what is needed are compositions and methods that are amenable tohigh throughput technology that can accurately provide real timeinformation regarding the interactions between potentially allintracellular components at the same time.

SUMMARY

The present invention is related to the field of intracellularbiochemistry. For example, the spatial proximity of intracellularcomponents may be related to their ability to cooperate in intracellularbiochemical reactions. In some embodiments, the present inventioncontemplates a variety of nucleic acid barcoded binding partners capableof determining the spatial proximity of intracellular components asdetermined by ligation of their respective nucleotide barcodes. As such,an intracellular component contact map may be constructed to fingerprintspecific physiological and/or pharmacological intracellular conditions.

In one embodiment, the present invention contemplates a compositioncomprising a binding partner attached to a unique nucleotide barcodesequence. In one embodiment, the binding partner is selected from thegroup consisting of an antibody, a locked nucleic acid, a receptor, aderivatized bead, a biological cell, and a small organic molecule. Inone embodiment, the unique nucleotide barcode sequence comprises aspecific primer pair sequence. In one embodiment, the unique nucleotidebarcode sequence comprises a first stand and a second strand. In oneembodiment, the first strand comprises a ‘3 primer sequence and a 5’primer sequence. In one embodiment, the first strand comprises a firstnucleic acid sequence and a linker molecule. In one embodiment, thesecond strand comprises a second nucleic acid sequence, wherein saidsecond nucleic acid sequence is complementary to said first nucleic acidsequence. In one embodiment, the linker molecule attaches said firststrand to said binding partner.

In one embodiment, the present invention contemplates a compositioncomprising a binding partner attached to a forked adapter molecule. Inone embodiment, the binding partner is selected from the groupconsisting of an antibody, a locked nucleic acid, a receptor, aderivatized bead, a biological cell, and a small organic molecule. Inone embodiment, the forked adapter molecule comprises a uniquenucleotide barcode sequence. In one embodiment, the forked adaptermolecule comprises a first stand and a second strand. In one embodiment,the first strand comprises a first nucleic acid sequence and a linkermolecule. In one embodiment, the second strand comprises a secondnucleic acid sequence, wherein said second nucleic acid sequence iscomplementary to said first nucleic acid sequence. In one embodiment,the linker molecule attaches said first strand to said binding partner.

In one embodiment, the present invention contemplates a compositioncomprising a nucleic acid sequence having a 3′-5′ first strand and a5′-3′ second strand, wherein said first strand 3′ end is attached to afirst binding partner and said second strand 3′ end is attached to asecond binding partner. In one embodiment, the first strand 3′ endfurther comprises a first primer. In one embodiment, the first strand 5′end further comprises a second primer. In one embodiment, the nucleicacid sequence comprises an asymmetric nucleotide barcode sequence. Inone embodiment, the first binding partner is selected from the groupconsisting of an antibody, a locked nucleic acid, a receptor, aderivatized bead, a biological cell, and a small organic molecule. Inone embodiment, the second binding partner is selected from the groupconsisting of an antibody, a locked nucleic acid, a receptor, aderivatized bead, a biological cell, and a small organic molecule.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a first binding partner having affinity fora first intracellular component, wherein said first binding partner isattached to a first unique nucleotide barcode sequence; ii) a secondbinding partner having affinity for a second intracellular component,wherein said second binding partner is attached to a second uniquenucleotide barcode sequence; iii) a biological sample comprising saidfirst and second intracellular components; and b) contacting said firstand second binding partners with said sample under conditions such thatan asymmetric nucleotide barcode sequence is created. In one embodiment,the contacting further comprises binding said first binding partner tosaid first intracellular component. In one embodiment, the contactingfurther comprises binding said second binding partner to said secondintracellular component. In one embodiment, the first unique nucleotidebarcode comprises double stranded deoxyribonucleic acid. In oneembodiment, the second unique nucleotide barcode comprises doublestranded deoxyribonucleic acid. In one embodiment, the contactingfurther comprises ligating said first double stranded unique nucleotidebarcode and said second double stranded unique nucleotide barcode.

In one embodiment, the first single stranded deoxyribonucleic acidcomprises a unique nucleotide barcode and a self-complementary sequenceat the 3′ end. In one embodiment, the second deoxyribonucleic acidcomprises a unique nucleotide barcode and the same self-complementarysequence at the 3′ end. In one embodiment, the single-strandeddeoxyribonucleotides contain a uracil 5′ to the unique nucleotidebarcodes. In one embodiment, the contacting further comprises annealingsaid first single stranded nucleotide and said second single strandednucleotide via the common self-complementary sequence at the 3′-endsfollowed by bi-directional primer extension of each 3′-end, therebycreating a double stranded deoxyribonucleic acid sequence that comprisesthe asymmetric nucleotide barcode sequence. In one embodiment, theprimers are extended by a DNA-dependent DNA polymerase. In oneembodiment, the primers are extended by a reverse transcriptase. In oneembodiment, the method further comprises cleaving said double-strandedasymmetric nucleotide barcode sequence formed by primer extension offthe two binding partners, followed by ligation to suitable adaptersthereby forming amplicons that can be PCR amplified. In one embodiment,cleavage is carried out by enzymatic excision of the uracil-containingnucleoside in the double-stranded product of the primer extensionfollowed by enzymatic cleavage of the phosphodiester bond on thecomplementary strand directly opposite to the gap left after excision ofthe uracil-containing nucleoside. Enzymes that catalyze the uracilexcision and phosphodiester cleavage are well know to those skilled inthe art.

In one embodiment, the first unique nucleotide barcode comprises singlestranded deoxyribonucleic acid. In one embodiment, the second uniquenucleotide barcode comprises single stranded deoxyribonucleic acid. Inone embodiment, the first single stranded nucleotide comprises a firstsequence complementary to a first primer. In one embodiment, the secondunique nucleotide barcode comprises a second sequence complementary to asecond primer, wherein the second sequence is palindromic to the firstsequence. In one embodiment, the contacting further comprises linkingsaid first single stranded nucleotide and said second single strandednucleotide, thereby creating a double stranded deoxyribonucleic acidsequence. In one embodiment, the double stranded deoxyribonucleic acidsequence comprises the asymmetric nucleotide barcode sequence. In oneembodiment, the linking comprises primer extension. In one embodiment,the primer extension comprises a reverse transcriptase. In oneembodiment, the method further comprises amplifying said asymmetricnucleotide barcode sequence, thereby forming amplicons. In oneembodiment, the method further comprises sequencing said amplicons,thereby identifying said ligated first and second unique nucleotidebarcode sequences. In one embodiment, the method further comprisesconstructing an intracellular component contact map by determiningjuxtaposed intracellular components from said identified ligated firstand second unique nucleotide barcode sequences. In one embodiment, thecontact map comprises a heat map.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a first single stranded nucleotide strandcomprising a 3′ forked end, wherein said 3′ forked end comprises a firstlinker molecule; ii) a second single stranded nucleotide strandcomprising a 5′ forked end, wherein said second strand is complementaryto said first strand; and iii) a binding partner comprising a secondlinker molecule, wherein said second linker molecule is capable ofconjugating with said first linker molecule; b) contacting said bindingpartner with said first single stranded nucleotide strand underconditions such that said first linker molecule conjugates with saidsecond linker molecule; and c) hybridizing said second strand with saidfirst strand. In one embodiment, the first linker molecule comprises5-HyNic. In one embodiment, the second linker molecule comprises S-4FB.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a first single stranded nucleotide strandcomprising a 3′ forked end, wherein said 3′ forked end comprises a firstlinker molecule; ii) a second single stranded nucleotide strandcomprising a 5′ forked end, wherein said second strand is complementaryto said first strand; and iii) a binding partner comprising a secondlinker molecule, wherein said second linker molecule is capable ofconjugating with said first linker molecule; c) hybridizing said secondstrand with said first strand to create a forked adapter molecule; andb) contacting said forked adapter molecule with said binding partnerunder conditions such that said first linker molecule conjugates withsaid second linker molecule. In one embodiment, the first linkermolecule comprises 5-HyNic. In one embodiment, the second linkermolecule comprises S-4FB.

In one embodiment, the present invention contemplates a kit, comprising:a) a first container comprising a plurality binding partners, whereineach said binding partner is attached to a different forked adaptermolecule; b) a second container comprising a solution capable of fixinga biological sample; c) a third container comprising buffers andreagents capable of supporting binding of said binding partner tointracellular components of said fixed biological sample; and d)instructions describing how to identify said intracellular componentsbound to said binding partners. In one embodiment, the forked adaptermolecule comprises a unique nucleotide barcode sequence. In oneembodiment, the binding partners are selected from the group consistingof antibodies, locked nucleic acids, intracellular receptors, and smallorganic molecules. In one embodiment, the instructions describeconstruction of an intracellular component contact map. In oneembodiment, the contact map comprises a heat map.

DEFINITIONS

The term “binding partner” as used herein, refers to any molecule havinga specific affinity for a particular intracellular component. Suchmolecules include, but are not limited to, antibodies, locked nucleicacids, receptors, biological cells, derivatized beads, or small organicmolecules. Alternatively, a binding partner may also include, atargeting partner as presently understood in the art.

The term “locked nucleic acid” as used herein, refers to any bicyclicnucleic acid where a ribonucleoside is linked between the 2′-oxygen andthe 4′-carbon atoms with a methylene unit.

The term “forked adapter molecule” as used herein, refers to any duplexnucleic acid having a first and second strand, wherein the strandsencode a unique nucleic acid barcode sequence. The first strandcomprising a 3′ forked end capable of conjugation with a bindingpartner. As such, the second strand comprises a 5′ forked end that iscomplementary to the first strand.

The term “unique nucleic acid barcode sequence” as used herein, refersto a specific nucleic acid sequence encoded within a forked adaptermolecule capable of providing an unambiguous identification of aspecific intracellular component.

The term “linker molecule” as used herein, refers to any organiccompound comprising a plurality of reactive sites, wherein a firstreactive site is capable of conjugation with either a forked adaptersequence or a binding partner and a second reactive site is capable ofconjugation with another linker molecule.

The term “asymmetric nucleotide barcode sequence” as used herein, refersto a joining (i.e., for example, by ligation) of two unique nucleotidebarcode sequences. Sequencing of the asymmetric nucleotide barcodesequence provides information related to the proximal location ofspecific intracellular components.

The term “intracellular component” as used herein, refers to anybiological structure in contact with the cytoplasm of a cell. Forexample, such components may comprise, proteins, enzymes, lipids,nucleic acids, riboproteins, or carbohydrates. Further, these componentsmay represent intracellular organelles including, but not limited to,Golgi bodies, endoplasmic reticulum, nuclear material, ribosomes,mitochondria etc.

The term “close proximity” as used herein, refers to a distance betweentwo intracellular components wherein an interaction between the twocomponents would be expected. Such a distance may range betweenapproximately 0.5 nm-100 nm. Preferably, such a distance may rangebetween approximately 5-50 nm. More preferably, such a distance mayrange between approximately 10-30 nm. Most preferably, such a distancemay range between approximately 15-20 nm.

The term “contact map” as used herein, refers to any presentation ofintracellular component organization as defined by spatial proximity.Such contact maps represent a spatio-functional status of a cell basedupon the current physiological and/or biochemical state as reflected bythe presence of asymmetric nucleotide barcode amplicons. For example,one representation of a contact map is a heat map that presents a visualrepresentation of an array of sequenced asymmetric nucleotide barcodes.The physiological and/or biochemical state of cell may be altered by,for example, changes in cell cycle statue, changes in temperature,changes in pH, drug exposure, toxin exposure. Any change in thephysiological and/or biochemical state of a cell would be expected tochange the contact map as determined by changes in the identifiedasymmetric nucleotide barcode amplicon concentrations.

The term “heatmap”, as used herein, refers to any graphicalrepresentation of data where the values taken by a variable in atwo-dimensional map are represented as colors. Heat maps have beenwidely used to represent the level of mRNA expression of many genesacross a number of comparable samples (e.g. cells in different states,samples from different patients) as obtained from DNA microarrays.

The term “attached” as used herein, refers to any interaction between amedium or carrier and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.

The term “medium” as used herein, refers to any material, or combinationof materials, which serve as a carrier or vehicle for delivering of adrug to an intracellular component. For all practical purposes,therefore, the term “medium” is considered synonymous with the term“carrier”.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering” a drug or compound, as usedherein, refers to any method of providing a drug or compound to abiological cell or tissue such that the drug or compound has itsintended effect on the biological cell or tissue. Such biological cellsor tissues may be derived from a patient.

The term “patient”, as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.” A patient may comprise any age of a human or non-humananimal and therefore includes both adult and juveniles (i.e., children).It is not intended that the term “patient” connote a need for medicaltreatment, therefore, a patient may voluntarily or involuntarily be partof experimentation whether clinical or in support of basic sciencestudies.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an compound that has a highaffinity for a receptor will provide greater efficacy in preventing thereceptor from interacting with its natural ligands, than a compound witha low affinity.

The term “derived from” as used herein, refers to the source of acompound (i.e., for example, a drug or toxin) or sequence (i.e., forexample, amino acid or nucleic acid). In one respect, a compound orsequence may be derived from an organism or particular species. Inanother respect, a compound or sequence may be derived from a largercomplex or sequence.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances that consist of amino acidresidues joined by peptide bonds, contain the elements carbon, hydrogen,nitrogen, oxygen, usually sulfur (i.e., for example, binding ligands,hormones, enzymes, antibodies, intracellular structural components). Ingeneral, a protein comprises amino acids having an order of magnitudewithin the hundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to “apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The term “an isolated nucleic acid”, as used herein, refers to anynucleic acid molecule that has been removed from its natural state(e.g., removed from a cell and is, in a preferred embodiment, free ofother genomic nucleic acid).

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

The term “antibody” refers to immunoglobulin evoked in animals by animmunogen (antigen). It is desired that the antibody demonstratesspecificity to epitopes contained in the immunogen. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., for example, an antigenic determinant orepitope) on a protein; in other words an antibody is recognizing andbinding to a specific protein structure rather than to proteins ingeneral. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabelled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

As used herein, the term “antisense” is used in reference to RNAsequences which are complementary to a specific RNA sequence (e.g.,mRNA). Antisense RNA may be produced by any method, including synthesisby splicing the gene(s) of interest in a reverse orientation to a viralpromoter which permits the synthesis of a coding strand. Once introducedinto a cell, this transcribed strand combines with natural mRNA producedby the cell to form duplexes. These duplexes then block either thefurther transcription of the mRNA or its translation. In this manner,mutant phenotypes may be generated. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. The designation (−) (i.e., “negative”) is sometimes usedin reference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL) which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA (in solution or bound to asolid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

The term “functionally equivalent codon”, as used herein, refers todifferent codons that encode the same amino acid. This phenomenon isoften referred to as “degeneracy” of the genetic code. For example, sixdifferent codons encode the amino acid arginine.

A “variant” of a protein is defined as an amino acid sequence whichdiffers by one or more amino acids from a polypeptide sequence or anyhomolog of the polypeptide sequence. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties, e.g., replacement of leucine with isoleucine. Morerarely, a variant may have “nonconservative” changes, e.g., replacementof a glycine with a tryptophan. Similar minor variations may alsoinclude amino acid deletions or insertions (i.e., additions), or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs including,but not limited to, DNAStar® software.

A “variant” of a nucleotide is defined as a novel nucleotide sequencewhich differs from a reference oligonucleotide by having deletions,insertions and substitutions. These may be detected using a variety ofmethods (e.g., sequencing, hybridization assays etc.).

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An “insertion” or “addition” is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues.

A “substitution” results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

The term “derivative” as used herein, refers to any chemicalmodification of a nucleic acid or an amino acid. Illustrative of suchmodifications would be replacement of hydrogen by an alkyl, acyl, oramino group. For example, a nucleic acid derivative would encode apolypeptide which retains essential biological characteristics.

The term “biologically active” refers to any molecule having structural,regulatory or biochemical functions.

The term “immunologically active” defines the capability of a natural,recombinant or synthetic peptide, or any oligopeptide thereof, to inducea specific immune response in appropriate animals or cells and/or tobind with specific antibodies.

The term “antigenic determinant” as used herein refers to that portionof a molecule that is recognized by a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms “immunogen,” “antigen,” “immunogenic” and “antigenic” refer toany substance capable of generating antibodies when introduced into ananimal. By definition, an immunogen must contain at least one epitope(the specific biochemical unit capable of causing an immune response),and generally contains many more. Proteins are most frequently used asimmunogens, but lipid and nucleic acid moieties complexed with proteinsmay also act as immunogens. The latter complexes are often useful whensmaller molecules with few epitopes do not stimulate a satisfactoryimmune response by themselves.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “C-A-G-T,” iscomplementary to the sequence “G-T-C-A.” Complementarity can be“partial” or “total.” “Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The terms “homology” and “homologous” as used herein in reference tonucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). A nucleotide sequence which is partiallycomplementary, i.e., “substantially homologous,” to a nucleic acidsequence is one that at least partially inhibits a completelycomplementary sequence from hybridizing to a target nucleic acidsequence. The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous sequence to atarget sequence under conditions of low stringency. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementarity(e.g., less than about 30% identity); in the absence of non-specificbinding the probe will not hybridize to the second non-complementarytarget.

The terms “homology” and “homologous” as used herein in reference toamino acid sequences refer to the degree of identity of the primarystructure between two amino acid sequences. Such a degree of identitymay be directed a portion of each amino acid sequence, or to the entirelength of the amino acid sequence. Two or more amino acid sequences thatare “substantially homologous” may have at least 50% identity,preferably at least 75% identity, more preferably at least 85% identity,most preferably at least 95%, or 100% identity.

Low stringency conditions comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent {50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)} and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed. Numerous equivalent conditions mayalso be employed to comprise low stringency conditions; factors such asthe length and nature (DNA, RNA, base composition) of the probe andnature of the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol), as well as components of the hybridizationsolution may be varied to generate conditions of low stringencyhybridization different from, but equivalent to, the above listedconditions. In addition, conditions which promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.) may also be used.

As used herein, the term “hybridizing”, “hybridize”, “hybridization”,“annealing”, or “anneal” are used interchangeably in reference to thepairing of complementary nucleic acids using any process by which astrand of nucleic acid joins with a complementary strand through basepairing to form a hybridization complex. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the melting temperature (T_(m)) of the formed hybrid, and theG:C ratio within the nucleic acids.

As used herein the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bounds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. As indicated by standard references, asimple estimate of the T_(m) value may be calculated by the equation:T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at1M NaCl. Anderson et al., “Quantitative Filter Hybridization” In:Nucleic Acid Hybridization (1985). More sophisticated computations takestructural, as well as sequence characteristics, into account for thecalculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. “Stringency” typically occurs in a rangefrom about T_(m) to about 20° C. to 25° C. below T_(m). A “stringenthybridization” can be used to identify or detect identicalpolynucleotide sequences or to identify or detect similar or relatedpolynucleotide sequences. Alternatively, when conditions of “weak” or“low” stringency are used hybridization may occur with nucleic acidsthat are derived from organisms that are genetically diverse (i.e., forexample, the frequency of complementary sequences is usually low betweensuch organisms).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of a targetsequence of interest. In contrast, “background template” is used inreference to nucleic acid other than sample template which may or maynot be present in a sample. Background template is most ofteninadvertent. It may be the result of carryover, or it may be due to thepresence of nucleic acid contaminants sought to be purified away fromthe sample. For example, nucleic acids from organisms other than thoseto be detected may be present as background in a test sample.

“Amplification” is defined as the production of additional copies of anucleic acid sequence (i.e., for example, amplicons) and is generallycarried out using polymerase chain reaction. Dieffenbach C. W. and G. S.Dveksler (1995) In: PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202,herein incorporated by reference, which describe a method for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of two oligonucleotide primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”. With PCR, it is possible to amplify a single copy ofa specific target sequence in genomic DNA to a level detectable byseveral different methodologies (e.g., hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxy-ribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “self-complementary sequence” refers to a firstnucleic acid sequence on a first oligonucleotide, wherein a secondoligonucleotide comprises a second nucleic acid sequence in reverseorder of the first nucleic acid. In this manner, the first and secondnucleic acid sequences are complementary and may hybridize, therebyannealing the first and second oligonucleotides.

As used herein, the term “ligate”, “ligating” or “ligation” refers toany method or composition wherein two different double strandednucleotides have been joined into a single oligonucleotide strand byachemic. Usually, a ligase enzyme facilitates the joining process.

As used herein, the term “linking” or “linked” refers to any method orcomposition wherein two different molecules have been joined by achemical reaction and/or enzymatic activity.

As used herein, the term “primer extension” refers to any method whereintwo different oligonucleotides become linked by an overlap of theirrespective terminal complementary primer sequences (i.e., for example, a3′ terminus). Such linking can be followed by an ezymatic extension ofboth termini using the other oligonucleotide as a templeate. Theezymatic extension may be performed by enzymes including, but notlimited to, DNA-dependent DNA polymerases and/or reverse transcriptases.

As used herein, the term “probe” comprises an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The terms “5′ ends” and “3′ ends” as used herein, refer to the terminiof oligonucleotides because mononucleotides are reacted to makeoligonucleotides in a manner such that the 5′ phosphate of onemononucleotide pentose ring is attached to the 3′ oxygen of its neighborin one direction via a phosphodiester linkage. Therefore, an end of anoligonucleotide is referred to as the “5′ end” if its 5′ phosphate isnot linked to the 3′ oxygen of a mononucleotide pentose ring. An end ofan oligonucleotide is referred to as the “3′ end” if its 3′ oxygen isnot linked to a 5′ phosphate of another mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene, i.e. the nucleic acid sequence which encodes agene product. The coding region may be present in a cDNA, genomic DNA orRNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in adjacent to the codingregion of the gene, if needed, to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. Transcriptional control signals ineukaryotes comprise “promoter” and “enhancer” elements. Promoters andenhancers consist of short arrays of DNA sequences that interactspecifically with cellular proteins involved in transcription. Maniatis,T. et al., Science 236:1237 (1987). Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inplant, yeast, insect and mammalian cells and viruses (analogous controlelements, i.e., promoters, are also found in prokaryotes). The selectionof a particular promoter and enhancer depends on what cell type is to beused to express the protein of interest. The presence of “splicingsignals” on an expression vector often results in higher levels ofexpression of the recombinant transcript. Splicing signals mediate theremoval of introns from the primary RNA transcript and consist of asplice donor and acceptor site. Sambrook, J. et al., In: MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor laboratoryPress, New York (1989) pp. 16.7-16.8. A commonly used splice donor andacceptor site is the splice junction from the 16S RNA of SV40.

The term “poly A site” or “poly A sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. A heterologous poly A signal isone which is isolated from one gene and placed 3′ of another gene.Efficient expression of recombinant DNA sequences in eukaryotic cellsinvolves expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length.

The term “transfection” or “transfected” refers to the introduction offoreign DNA into a cell.

As used herein, the terms “nucleic acid molecule encoding”, “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

The term “Southern blot” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size, followed bytransfer and immobilization of the DNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized DNA is thenprobed with a labeled oligodeoxyribonucleotide probe or DNA probe todetect DNA species complementary to the probe used. The DNA may becleaved with restriction enzymes prior to electrophoresis. Followingelectrophoresis, the DNA may be partially depurinated and denaturedprior to or during transfer to the solid support. Southern blots are astandard tool of molecular biologists. J. Sambrook et al. (1989) In:Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp9.31-9.58.

The term “Northern blot” as used herein refers to the analysis of RNA byelectrophoresis of RNA on agarose gels to fractionate the RNA accordingto size followed by transfer of the RNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized RNA is thenprobed with a labeled oligodeoxyribonucleotide probe or DNA probe todetect RNA species complementary to the probe used. Northern blots are astandard tool of molecular biologists. J. Sambrook, J. et al. (1989)supra, pp 7.39-7.52.

The term “reverse Northern blot” as used herein refers to the analysisof DNA by electrophoresis of DNA on agarose gels to fractionate the DNAon the basis of size followed by transfer of the fractionated DNA fromthe gel to a solid support, such as nitrocellulose or a nylon membrane.The immobilized DNA is then probed with a labeled oligoribonuclotideprobe or RNA probe to detect DNA species complementary to the ribo probeused.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequencecoding for RNA or a protein. In contrast, “regulatory genes” arestructural genes which encode products which control the expression ofother genes (e.g., transcription factors).

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene and includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences whichare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ non-translated sequences. The sequences which arelocated 3′ or downstream of the coding region and which are present onthe mRNA are referred to as 3′ non-translated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene which are transcribed intoheterogeneous nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide. In addition to containing introns, genomic forms of a genemay also include sequences located on both the 5′ and 3′ end of thesequences which are present on the RNA transcript. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “label” or “detectable label” are used herein, to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein,texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include, but are not limited to,U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241 (all herein incorporated by reference). Thelabels contemplated in the present invention may be detected by manymethods. For example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting, the reaction product produced by the action of the enzyme onthe substrate, and calorimetric labels are detected by simplyvisualizing the colored label.

The term “binding” as used herein, refers to any interaction between atleast two compounds. Binding may be reversible or irreversible. Suchbinding may be, but is not limited to, non-covalent binding, covalentbonding, ionic bonding, Van de Waal forces or friction, and the like.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 illustrates one embodiment of the invention.

FIG. 1A: A SNAPPLE probe comprising a binding partner including, but notlimited, to an antibody or a locked nucleic acid (LNA) linked to aforked adapter molecule having a unique nucleotide barcode sequence.

FIG. 1B: Juxtaposition of two SNAPPLE probes in a sample, wherein thecorresponding unique nucleotide barcode sequences are brought into closeproximity and

FIG. 1C: Ligation of the unique nucleotide barcode sequences between twojuxtaposed probes to form an asymmetric nucleotide barcode sequence.

FIG. 2 illustrates one embodiment of the invention.

FIG. 2A: An asymmetric nucleotide barcode sequence capable ofamplification using standard Illumina primers.

FIG. 2B: A symmetric nucleotide barcode sequence forming a hairpinwherein amplification is suppressed.

FIG. 2C: An unligated unique nucleotide barcode sequence that cannot beamplified.

FIG. 3 illustrates three possible embodiments of SNAPPLE probes whereineach probe (e.g., A′, B′, and C′) has specific affinity to a uniqueintracellular component (e.g., Protein A, Protein B, and Protein C) andeach probe is conjugated to a unique nucleotide barcode sequence (e.g.A″, B″, and C″).

FIG. 4 illustrates the spatial localization of representativeintracellular components of FIG. 3, following a fixation step.

FIG. 5 illustrates the binding of the SNAPPLE probes to the fixedintracellular components of FIG. 4. Note the close proximity of SNAPPLEprobe A′ and B′ binding to co-located Protein A and Protein B,respectively.

FIG. 6 illustrates the blunt end ligation of the unique nucleotidebarcodes of the SNAPPLE probes after attachment to their respectiveintracellular components. Note that, in this case, an asymmetricnucleotide barcode sequence is formed as a junction between the twoproximal SNAPPLE probes attached to Protein A and Protein B.

FIG. 7 illustrates one embodiment of two ligated SNAPPLE probescomprising an asymmetric nucleic acid barcode sequence (area betweendashed lines). Arrows: Forked end adapter.

FIG. 8 illustrates one embodiment of a SNAPPLE probe comprising abinding partner (BP) conjugated to a unique nucleotide barcode sequence(UNB) having a forked adaptor configuration. In this illustration, afirst end of the forked adapter (solid arrow) is linked to the bindingpartner, while the second end of the forked adapter (dashed arrow) isfree.

FIG. 9 presents a close up view of one embodiment of a juxtaposition oftwo UNBs from two different SNAPPLE probes before ligation.

FIG. 10 presents a close up view of one embodiment of a ligatedasymmetric nucleotide barcode sequence comprising UNBs from twodifferent SNAPPLE probes, wherein a 3′ and 5′ primer pair (see arrows)have been hybridized.

FIG. 11 presents a schematic of the amplification of the ligatedasymmetric barcode using a specific primer pair (arrows), therebyresulting in amplicons of the joined UNBs.

FIG. 12 presents one embodiment for making a SNAPPLE probe byconjugating a first linker molecule (LM1) to a binding partner (BP).

FIG. 13 presents one embodiment for making a SNAPPLE probe byconjugating a second linker molecule (LM2) to a first single strandedforked adapter molecule (ssFAM1) attached to a first single strandedunique nucleotide barcode sequence (ssUNB1).

FIG. 14 presents one embodiment for making a SNAPPLE probe byhybridizing a LM2-ssFAM1-ssUNB1 to a second single stranded forkedadapter molecule (ssFAM2) having a complementary single stranded uniquenucleotide sequence (ssUNB1c) to create a forked adapter molecule (FAM).

FIG. 15 presents one embodiment for making a SNAPPLE probe byhybridizing an ssFAM1 and an ssFAM1c to form a forked adapter molecule(FAM).

FIG. 16 presents one embodiment for making a SNAPPLE probe byconjugating a second linker partner (LM2) to a forked adapter molecule(FAM) to create an LM2-FAM molecule.

FIG. 17 presents one embodiment for making a SNAPPLE probe byconjugating a binding partner attached to a first linker molecule(BP-LM1) with a forked adapter molecule attached to a second linkermolecule (LM2-FAM) to form a binding partner-forked adapter moleculecomplex (BP-FAM).

FIG. 18 presents one embodiment of a SNAPPLE probe comprising a lockednucleic acid (LNA) binding partner.

FIG. 19 presents one embodiment of a messenger RNA having multipleSNAPPLE probe binding sites.

FIG. 20 presents several embodiments of locked nucleic acids:

FIG. 20A: 5-Me-Bz-C LNA

FIG. 20B: Bz-A LNA

FIG. 20C: dmf-G LNA

FIG. 20D: T LNA

DETAILED DESCRIPTION

The present invention is related to the field of intracellularbiochemistry. For example, the spatial proximity of intracellularcomponents may be related to their ability to cooperate in intracellularbiochemical reactions. In some embodiments, the present inventioncontemplates a variety of nucleic acid barcoded binding partners capableof determining the spatial proximity of intracellular components asdetermined by ligation of their respective nucleotide barcodes. As such,an intracellular component contact map may be constructed to fingerprintspecific physiological and/or pharmacological intracellular conditions.

I. Protein Proximity Detection by DNA Ligation

The use of nucleotide barcodes have been used to identify proteins. Forexample, one method is termed proximity ligation. This method utilizes anucleotide affinity probe having two recognition sites for a targetmolecule (i.e., a protein), followed by amplification to provide adetection signal. Fredriksson et al., “Protein detection DNA usingproximity-dependent ligation assays” Nature Biotechnology 20:473-477(2002). A homodimer of the platelet-derived growth factor B-chain(PDGF-BB), was recently studied. DNA aptamers were used as affinityprobes that were obtained through a process of in vitro selection foraffinity to PDGF-BB. The selected DNA aptamers were extended withadditional nucleotide sequence elements at either the 5′ or the 3′ end,forming a proximity probe pair. Green et al., “Inhibitory DNA ligands toplatelet-derived growth factor B-chain” Biochemistry 45:14413-14424(1996). When two of these probes bind to the same PDGF-BB molecule,their respective sequence extensions hybridize together provided aconnector oligonucleotide has been added. This connector oligonucleotidefacilitates an enzymatic DNA ligation of the two sequence extensions.The ligation products can then be replicated by nucleic acidamplification through PCR, while unreacted probes remain silent. Insummary, this protocol uses two DNA aptamers to identify and localize asingle protein. This in vitro DNA amplification technique is applicableto the acquisition of genomic expression information for detection ofspecific proteins, and not the interaction of a first protein with asecond protein having zeptomolar sensitivity (i.e., 40×10⁻²¹ mol).

Other proximity ligation-based protein detection procedures may detect aprotein complex via unique nucleic-acid identifiers and subsequentquantification by real-time PCR. Fredriksson et al., “Multiplexedprotein detection by proximity ligation for cancer biomarker validation”Nature Methods 4:327-329 (2007). This technology (multiplexed proximityligation) uses a pair of proximity probes, wherein each probe iscomposed of an antibody linked to an oligonucleotide, wherein bothantibodies have affinity for the same protein. Once both antibodies arebound to the protein, a connecting oligonucleotide is then hybridized tothe linked nucleotide of both probes. This provides an “oligonucleotidebridge” that enables an enzymatic ligation joining the 3′ end of thefirst probe with the 5′-end of the second probe. This ligation forms aunique target reporter amplicon containing a specific molecular barcode.Hardenbol et al., Nat. Biotechnol. 21:673-678 (2003). These molecularbarcodes serve as primer sites, of which some are universal for allprotein analytes, whereas others are target-specific. The amplicons areanalyzed by real-time PCR thereby generating quantitativeprotein-abundance data. The assay reporter signal is dependent on aproximal and dual recognition of each target analyte. These assays usehigh concentrations of proximity probes to promote target binding andensure a wide dynamic range while maintaining low levels of backgroundligation events. The background noise in proximity ligation is derivedfrom two main sources: first, proximity probes nonspecifically bindingto each other, and second, the connector oligonucleotide binding to twofreely diffusing probes, enabling ligation. In general, the workflow ofmultiplexed proximity ligation assays conceptually resembles that ofcDNA synthesis, but for targeted proteins only.

Adaptations of proximity ligation methods having fluorescence baseddetection methods, have examined the subcellular localization ofprotein-protein interactions (proximity ligation in situ assay; P-LISA).In one approach, proximity probes having oligonucleotides attached toantibodies were targeted to two different proteins. The first probe'soligonucleotide has a tag sequence that is complementary to afluorescent oligonucleotide and a primer sequence. The second probe isnonpriming due to a blocking 2′-O-methyl RNA derivative partner. Whenbrought into close proximity, the juxtaposed probe oligonucleotides arestabilized by hybridizing to a connector oligonucleotide, wherein theconnector oligonucleotide forms a circular DNA strand. The DNA circle,in turn, serves as a template for localized rolling-circle amplification(RCA) that generates amplicons of the first probe's oligonucleotide tagsequence. When the first probe oligonucleotide tag amplicons arehybridized with a fluorescent oligonucleotide, the intracellularlocation of the interacting protein pair may be visualized as coloredspots. This method has been used to verify protein-protein interactionsbetween endogenous Myc and Max oncogenic transcription factors inresponse to interferon-c (IFN-c) signaling and low-molecular-weightinhibitors. Soderberg et al., “Direct observation of individualendogenous protein complexes in situ by proximity ligation” NatureMethods 3:995-1000 (2006). The oligonucleotides on these P-LISAproximity probes, when brought into close proximity by binding adjacentproteins, serve as templates for the circularization of so-calledconnector oligonucleotides by enzymatic ligation. The circularized DNAstrands remain hybridized to the proximity probes. Upon addition of aphi29 DNA polymerase, the oligonucleotide of the first proximity probeserves as a primer for the RCA reaction, during which the processreleases the second probe's oligonucleotide from the DNA circle and isnot amplified. The second probe's oligonucleotide cannot serve as aprimer because this sequence has three mismatched, exonuclease-resistant2′-O-methyl RNA nucleotides at the 3′ end, which blocks polymeraseactivity. Consequently, the RCA reaction (˜1 hour) generates a randomlycoiled, single-stranded product complementary to the first probe'soligonucleotide, while the DNA circle is covalently linked to theantibody-antigen complex. Baner et al., “Signal amplification of padlockprobes by rolling circle replication” Nucleic Acids Res. 26:5073-5078(1998). This adduct product is then detected through hybridization of afluorescence-labeled oligonucleotide that is complementary to a tagsequence in the RCA product. While it is possible to increase the numberof proximity probes used in P-LISA to create larger circular amplifiableligation products to study multiprotein complexes, this method is notuseful to simultaneously detect and compare different multiproteincomplexes. In particular, P-LISA is not capable of generating anintracellular contact map, based upon simultaneous detection ofhundreds, if not thousands, of interacting cell components (i.e., forexample, proteins). This limitation is because P-LISA is dependent uponfluorescent detection signals for quantitation of assay results. On theother hand, the unique structure and method of using the presentlydisclosed SNAPPLE probes allow isolation and sequencing of ampliconsthat differentiate between hundreds, if not thousands, of interactingcell components.

Although it is not necessary to understand the mechanism of aninvention, it is believed that by varying the size and shape of bindingpartners (i.e., for example, full length intact antibodies or Fabfragments) and/or unique nucleotide barcode sequences on SNAPPLE probes,the methods herein can be used as a molecular ruler, thereby allowingmeasurements of distances between binding regions. For example, anaverage distance between binding regions may be approximately 30 nm,(i.e., for example, the size of the two antibodies and a uniquenucleotide barcode length). However, longer distances may also beenvisioned. Further, shorter distances could be used to improveresolution by limiting detection distances to just over 10 nm.

II. Sequencing-Mediated Numerical Analysis of Parallel ProximityLigation Events

In one embodiment, the present invention contemplates a method termedSequencing-Mediated Numerical Analysis of Parallel Proximity LigationEvents (SNAPPLE). Although it is not necessary to understand themechanism of an invention, it is believed that SNAPPLE represents onesequencing-based technique for identifying intracellular components thatare in proximity to one another. In one embodiment, the SNAPPLE methodfurther comprises high throughput analyses. For example, tens ofthousands of proteins, DNAs, RNAs, and small molecules can beindividually and simultaneously probed in a single sequencing run. Sucha method results in an intracellular component interaction contact map,yielding qualitative and quantitative data about all inter-componentproximity relations.

In one embodiment, the present invention contemplates a compositioncomprising a SNAPPLE probe. In one embodiment, the probe comprises abinding partner, capable of specific interaction with an intracellularcomponent. In one embodiment, the binding partner includes, but is notlimited to, an antibody, LNA, DNA, a binding ligand, a receptor, aderivatized bead, a biological cell, or a chemical partner (i.e., asmall organic molecule). In one embodiment, the probe further comprisesa forked oligonucleotide adapter molecule. In one embodiment, the forkedadapter molecule comprises a component-specific oligonucleotide barcode.See, FIG. 1A.

In one embodiment, the present invention contemplates a SNAPPLE methodcomprising a) contacting a sample with a fixative; b) incubating thefixed sample with a plurality of SNAPPLE probes; and c) blunt-endligating the probes. Although it is not necessary to understand themechanism of an invention, it is believed that when two different probesbind to their respective components that are spatially colocated, thecorresponding forked oligonucleotide adapter molecules may be ligated,thereby forming a junction comprising an asymmetric nucleotide barcode(i.e., wherein the junction comprises two different unique nucleotidebarcodes derived from two different SNAPPLE probes). See, FIG. 1B andFIG. 1C. In one embodiment, the method further comprises amplifying eachasymmetric nucleotide barcode with a different single primer pair. See,FIG. 2A. Although it is not necessary to understand the mechanism of aninvention, it is believed that unligated adapters will not be amplifiedand symmetric junctions lead to large hairpins that suppressamplification. See, FIG. 2B and FIG. 2C, respectively. In oneembodiment, the method further comprises sequencing the amplicons in,for example, an Illumina flowcell, to identify the unique nucleotidebarcodes that were ligated together. In one embodiment, the sequencingdata creates a ‘contact map’ identifying paired intracellular components(i.e., for example, those intracellular components that were in closeproximity). As such, many different binding partners may be usedtogether provided each binding partner is conjugated to a uniquenucleotide barcode sequence (i.e., thereby forming a different SNAPPLEprobe). For example, three different proteins (i.e., for example,Protein A, Protein B, and Protein C) may be assayed simultaneouslybecause each protein has a specific affinity for one of three differentbinding partners (i.e., for example, antibody A′, antibody B′, andantibody C′) wherein each binding partner is conjugated to a uniquenucleic acid barcode sequence (i.e., for example, sequence A″, sequenceB″, and sequence C″). See, FIG. 3.

Briefly, the SNAPPLE method comprises a fixation step that immobilizessuch representative proteins A & B in relation to their intracellularspatial localization that may reflect their biochemical functions. See,FIG. 4. The SNAPPLE method further comprises an incubation step, whereinthe SNAPPLE probes are introduced into the intracellular space underconditions such that they bind and/or attach to specific intracellularcomponents. In some situations, at least two SNAPPLE probes attachwithin close proximity. See, FIG. 5. The SNAPPLE method furthercomprises a ligation step, wherein the unique nucleotide barcodes on thejuxtaposed SNAPPLE probes are contacted with a ligase enzyme, therebyresulting in an asymmetric barcode sequence by blunt end ligation. See,FIG. 6. The SNAPPLE method further comprises washing, amplification, andisolation of the ligated SNAPPLE probes comprising an asymmetric barcodesequence. See, FIG. 7. The resulting configuration of the asymmetricbarcode sequence demonstrates the advantages of the “forked adaptor”design, such that after ligation, the two original barcodes display anunattached fork and an attached fork that facilitate attachment of theprimers in preparation for sequencing (see arrows).

A specific disadvantage of extant proximity ligation methods only allowa single probe to ligate to a single partner probe. While the abovedescribed ‘multiplex’ protein quantification method (i.e., Fredrikssonet al.) can utilize multiple probes, each probe is limited to ligateonly to a single partner probe. Secondly, multiplex techniques requiresthat each probe have two distinct binding sites for the target molecule,thereby increasing the formation of homodimeric ligations. Thesetechniques do not suggest designing probes such that any probe mayligate with any other probe with equal efficiency and still be able toread out the results via conventional nucleic acid sequencing analysis(i.e., for example, qPCR mediated sequencing).

For example, unlike multiplex proximity ligation techniques, someembodiments presented herein allow performance of qPCR after designing aset of N probes specific to N targets such that each oligo tag (i.e.,for example, a first UNB) can ligate to any and every other oligo tag(i.e., for example, a second, third, fourth, etc., UNB). Even when twodifferent short dsDNA's are conjugated to different probes and blunt endligated together, problems arise during conventional qPCR. If all theprobe dsDNA's hybridize to the same upstream primer A, then PCR fails togenerate amplicons because the resulting products will become hairpins,due to primer complemetarity at the single stranded cDNA strand ends.One the other hand, if each probe dsDNA sequence hybridizes to adifferent primer (i.e., primer A and primer B), the ligated dsDNAs canbe amplified with a mixture of primer A and primer B. This technique,however, requires twice as many probes and a much more complicatedprotocol generating many ligation products between A-A primed ligateddsDNAs and B-B primed ligated dsDNAs (i.e., homodimeric ligationdsDNAs). The dsDNAs having identical primers at each end do not respondefficiently to PCR, thereby decreaseing amplicon yield. It is believedthat these disadvantages cannot be eliminated by using ssDNA, overhangs,or linker oligos, which many of the proximity ligation protocolsadvocate. Although it is not necessary to understand the mechanism of aninvention, it is believed that for efficient qPCR of any heterodimericligated dsDNA the use of ssDNA, overhangs, or linker oligos generates anunacceptable amount of contaminating homodimeric PCR product artifactsas opposed to heterodimeric PCR products of interest.

In one embodiment, the present invention contemplates a forkedoligonucleotide sequence (i.e., for example, a forked adapter molecule)that is conjugated to a probe (i.e., for example, a binding partner)which is believed to overcome the homodimer qPCR problems describedabove. Although it is not necessary to understand the mechanism of aninvention, it is believed that by using a forked adapter molecule primerA/primer B, qPCR may be performed without the need for two probes pertarget, thereby vastly reducing the formation of homodimer PCR products.It is also believed that the hairpin PCR products that do form fromhomodimer PCR products, facilitate sequencing analysis because theysuppress same probe-same probe ligation product frequency.

A. Intracellular Component Complexes

In one embodiment, the present invention contemplates a pair of SNAPPLEprobes that are respectively linked to different antibodies, whereineach antibody comprises a specific affinity to an intracellularcomponent (i.e., for example, intracellular proteins). Although it isnot necessary to understand the mechanism of an invention, it isbelieved that if the two intracellular components tend to form acomplex, the proximal binding of the corresponding antibody-SNAPPLEprobe pair places the conjugated barcoded adapters into close proximity.It is further believed that this proximity facilitates ligation of theunique nucleotide barcodes, thereby creating a join specific to thejuxtaposed component pair (i.e., for example, an asymmetric ligatedbarcode). Thus, when the asymmetric ligated barcode is amplified, theamplified products comprise a specific nucleotide sequence thatidentifies the juxtaposed intracellular components (i.e., for example,an inter-protein complex). Other embodiments contemplate complexation ofprotein-RNA components or RNA-RNA components that may be identified andanalyzed in a similar manner.

B. Nucleic Acid Structure Determination

In one embodiment, a SNAPPLE probe comprises a plurality of bindingpartners, wherein the moieties attach to different regions on the sameintracellular component (i.e., for example, folded DNA or mRNA). See,FIG. 19. Although it is not necessary to understand the mechanism of aninvention, it is believed that these multi-targeted probes can be usedto approximately determine intracellular component folds orthree-dimensional (i.e., for example, tertiary or quaternary) structuresof an in vivo intracellular component. In one embodiment, theintracellular component comprises a protein having multipleconformations. In one embodiment, a multiple conformational proteincomprises a prion.

Such nucleic acid structure determinations may be accomplished bySNAPPLE probes comprising binding partners selected from commerciallyavailable chromatin-immunprecipiating antibodies (NimbleGen), forexample:

-   -   1. Core Histones & Modifications        -   Histone H2A Abcam ab18255        -   Histone H2B Abcam ab1790        -   Histone H3 Abcam ab1791 17277777, 15231737, 17220878,            17559302        -   Histone H4 Abcam ab7311        -   Histone H3 (me1-K4) Abcam ab8895 17277777, 17559302        -   Histone H3 (me2-K4) Upstate 07-030 17277777, 16980610,            15988478, 17559302        -   Histone H3 (me3-K4) Abcam ab8580 16604156, 17344414        -   Histone H3 (me1-K9) Abcam ab9045 15923188        -   Histone H3 (me3-K9) Abcam ab 1186 16751344, 17542650,            17604720        -   Histone H4 (me1-K20) Abcam ab9051 17512414        -   Histone H4 (me2-K20) Abcam ab1409 12397363        -   Histone H4 (me3-K20) Abcam ab9053 17512414        -   Histone H3 (me3-Lys27) Upstate 07-449 16751344, 16618801,            15231737, 17542650, 17339329, 17604720        -   Histone H3 (me1-K36) Abcam ab9048 17512414        -   Histone H3 (me3-K36) Abcam ab9050 16122420        -   Histone H3 (me1-K79) Abcam ab2886 17512414        -   Histone H3 (me2-K79) Abcam ab3594 17512414        -   Histone H3 (me3-K79) Abcam ab2621 16122420        -   Histone H4 (acetyl K5) Abcam ab1758 15292231        -   Histone H4 (acetyl K8) Abcam ab1760 15292231        -   Histone H4 (acetyl K12) Abcam ab1761        -   Histone H4 (acetyl K16) Abcam ab1762        -   Histone H4 (acetyl K5/8/12/16) Upstate 06-866 17277777,            17218097, 16980610, 17229572, 16914732, 17559302        -   Histone H3 (acetyl K9/14) Upstate 06-599 15988478, 17559302,            16473879, 16980610    -   2. Transcription Factors        -   AP-2α Santa Cruz sc-184X 17053090        -   AP-4 Santa Cruz sc-18595X 12391156        -   ATF-2 Santa Cruz sc-6233X 15226416        -   C/EBPβ Santa Cruz sc-150X 16914732        -   c-fos Santa Cruz sc-52X 14672732        -   c-jun Upstate 06-225 14764426        -   c-myb Santa Cruz sc-7874X 12377807        -   c-myc Santa Cruz sc-764X 16606705, 17568006        -   CREB Upstate 06-863 15194748        -   E2F1 (KH20/KH95) Upstate 05-379 17053090, 16606705        -   ERα Upstate 06-935 12897156        -   FOXA1 Abcam ab5089 15743813        -   GATA-1 Santa Cruz sc-265X 15456760        -   GR Santa Cruz sc-1004X 16914732        -   HIF1α Abcam ab2185        -   HNF-3γ Santa Cruz sc-5361X 15358835        -   HNF-4α Santa Cruz sc-8987X 12416993        -   HSF1 Stressgen SPA-901 14673135        -   Max Santa Cruz sc-765X 15226411        -   NF-kappaB p50 Upstate 06-886 12783888        -   NF-kappaB p65 Santa Cruz sc-8008X 14527995        -   Oct-3/4 Santa Cruz sc-8628X 17567999, 16751344        -   p53 Santa Cruz sc-6243X 15205322        -   RXR Santa Cruz sc-774X 16497728, 17229572, 16914732        -   Sp1 Upstate 07-124 15180995        -   Sp3 Upstate 07-107 15180995        -   Stat2 Santa Cruz sc-476X 14600148        -   Stat3 Santa Cruz sc-482X 14659888        -   Stat5α Santa Cruz se-1081X 14659888        -   SUZ12 Abcam ab12201 17542650, 16618801, 15231737, 16751344,            17604720        -   USF-1 Santa Cruz sc-8983X 15187018        -   VDR Santa Cruz sc-1008X 16613987, 17229572, 16914732        -   YY1 Santa Cruz sc-7341X 15326102    -   3. Chromatin Modifiers        -   BAF170 Santa Cruz sc-9744X 15314177        -   Brg-1 Santa Cruz sc-10768X 15314177        -   Brm Santa Cruz sc-6450X 15314177        -   CARM1 Upstate 07-080 14764426        -   CBP Santa Cruz sc-369X 16497728, 17218097        -   HDAC1 Upstate 06-720 15226416        -   HDAC2 Santa Cruz sc-7899X 12783888        -   LSD1 Abcam ab1772        -   mSin3A Santa Cruz sc-994X 15314177        -   NCoR Santa Cruz sc-8994X 15226416        -   p300 Santa Cruz sc-585X 17277777        -   PCAF Upstate 07-141 14764426        -   SUV39H1 Abeam ab12405        -   TRAP220 Santa Cruz sc-5334X 17277777    -   4. Pre-Initiation Complex        -   Pol II Santa Cruz sc-899X 16618801, 15231737, 17344414,            16606705        -   RNAPII, 8WG16 Covance MMS-126R 17277777, 15988478, 17604720        -   TAFII p250 (6B3) Santa Cruz sc-735X 17277777, 15988478        -   TFIIB Santa Cruz sc-274X 15247294        -   TFIID (TBP) Santa Cruz sc-273X 15280358        -   TFIIF Abcam ab4449        -   TFIIH p89 Santa Cruz sc-293X 11493692    -   5. DNA Methylation        -   5-methylcytidine Eurogentec BI-MECY-0500 16007088, 17128275        -   Dnmt1 Abcam ab5208 16357870        -   Dnmt3b Abcam ab2851 16357870        -   MBD1 Abcam ab3753 14633992        -   MBD3 Abcam ab3755 14633992        -   MeCP2 Abcam ab3752 14633992            II. SNAPPLE Probes

In one embodiment, the present invention contemplates a compositioncomprising a binding partner, wherein the partner is conjugated to anucleic acid sequence. In one embodiment, the nucleic acid sequencecomprises a forked adapter molecule. See, FIG. 8 (arrow). In oneembodiment, the forked adapter molecule is conjugated to the bindingpartner at a 3′ end. In one embodiment, the forked adapter moleculecomprises a nucleic acid sequence. In one embodiment, the forked adaptersequence comprises a linker molecule.

In one embodiment, the present invention contemplates a compositioncomprising two SNAPPLE probes attached by ligation between theirrespective unique nucleotide barcode sequences (UNBs). Such UNB ligationis facilitated because, after the binding partners are attached to theirrespective intracellular components, the UNBs are positioned in closeproximity. See, FIG. 9. After UNB ligation, specific primers may behybridized to the 3′ and 5′ ends of one UNB forked adapter moleculestrand. See, FIG. 10 (See arrows). After placement of the primers,quantitative polymerase chain reaction is performed upon the asymmetricbarcode sequence to form amplicons. See, FIG. 11. There are many ways toprocess the amplicon sequence readouts including, but not limited to: i)Illumina sequencing that may be capable of processing an unlimitednumber of amplicons; ii) microarray hybridizations that may be capableof processing hundreds of amplicons; and iii) Luminex Flow Sorting thatmay be capable of processing tens of amplicons.

In one embodiment, the present invention contemplates a method formaking a SNAPPLE probe comprising: providing a binding partner and afirst linker molecule. In one embodiment, the first linker moleculecomprises 5-HyNic. In one embodiment, the first linker molecule isconjugated to the binding partner to create a 5-HyNic conjugated bindingpartner (i.e., for example, an antibody). See, FIG. 12.

In one embodiment, the present invention contemplates a method formaking a SNAPPLE probe comprising: providing a first single strandedforked adapter molecule and a second linker molecule. In one embodiment,the second linker molecule comprises S-4FB. In one embodiment, thesecond linker is conjugated to the forked adapter molecule to create aS-4FB conjugated forked adapter, wherein the adapter is attached to afirst single stranded unique nucleotide barcode sequence. See, FIG. 13.In one embodiment, the first single stranded unique nucleotide barcodesequence is annealed with a second single stranded unique nucleotidebarcode sequence, wherein the second sequence is complementary to thefirst sequence. See, FIG. 14.

In one embodiment, the present invention contemplates a method formaking a SNAPPLE probe comprising: providing a first single strandedforked adapter molecule (ssFAM1) and a complementary first singlestranded forked adapter molecule (ssFAM1c). In one embodiment, themethod further comprising hybridizing the ssFAM1 and ssFAM1c, therebyforming a forked adapter molecule (FAM). See FIG. 15. In one embodiment,the FAM is conjugated to a second linker molecule (LM2; for example,S-4FB), to create a forked adapter molecule conjugated to a secondlinker molecule (LM2-FAM). See, FIG. 16.

In one embodiment, the present invention contemplates a method formaking a SNAPPLE probe comprising: providing a binding partnerconjugated to a first linker molecule and a forked adapter moleculeconjugated to a second linker molecule. In one embodiment, the first andsecond linker molecules are conjugated to form a binding partner-forkedadapter molecule complex (BP-FAM). See, FIG. 17.

A. Antibody Binding Partners

Antibody-based detection systems for specific antigens are useful forvarious molecular and cellular analyses and/or clinical diagnostics.Such systems are very flexible because antibody specificity can betailored to particular epitopes. For example, a number of antibodytechnologies include, but are not limited to, genetic engineering ofantibody molecules (M. J. Geisow, Trends Biotechnol. 10:75 (1992);production of catalytic antibodies (Lerner et al., Science 252:659(1991).); and bispecific antibodies (Bolhuis et al., J. Cell. Biochem.47:306 (1991). Further enhancement of antigen detection sensitivityshould facilitate the specific detection of rare antigens.

Antibody conjugation to oligonucleotides were reported to be useful inquantifying the presence of minute quantities of antigen (i.e., forexample, Immuno-PCR). Sano et al., Science 258:120-122 (1992). In thisearly work, a streptavidin-protein A chimera that possesses tight andspecific binding affinity both for biotin and immunoglobulin G was usedto attach a biotinylated DNA specifically to antigen-monoclonal antibodycomplexes that had been immobilized on microtiter plate wells. Then, asegment of the attached DNA was amplified by PCR. Analysis of the PCRproducts by agarose gel electrophoresis after staining with ethidiumbromide allowed as few as 580 antigen molecules (9.6×10⁻²² moles) to bereadily and reproducibly detected. Direct comparison with enzyme-linkedimmunosorbent assay with the use of a chimera-alkaline phosphataseconjugate demonstrated an enhancement in detection sensitivity (i.e.,for example, approximately×10⁵). Consequently, PCR amplification shouldimprove the sensitivity of any antigen detection system and, inprinciple, could be applied to the detection of single molecules.

The accuracy of such assays may be increased by using antibodies or DNAaptamers directed to a target protein having multiple epitopes followedby proximity ligation and PCR amplification of the ligation product.Fredriksson et al., Nature Biotechnology (2002); and Nature Methods(2007). It was speculated that multiple binding sites eliminatednon-specific binding by a single antibody. A similarproximity-ligation-based method was developed for localizing a singleprotein complex using a complex ligation/Rolling Circle Amplificationprotocol followed by fluorescence microscopy to detect the presence ofan encoded tag. Soderberg et. al., Nature Methods (2006). Pull-down ofgenomic targets using DNA may also be performed by related techniques.Dejardin et al., Cell (2009).

The development of SNAPPLE probes may be validated using the proteinsEGFR, Her-2, and c-Myc. It has been reported that EGFR and Her2 formcomplexes while EGFR/c and Myc do not form complexes in U2 osteosarcomacells. Soderberg et. al., Nature Methods (2006). Verification that theSNAPPLE biochemistry works may be performed by using: i) an EGFRprotein; ii) a Her-2 protein; iii) a c-Myc protein; and iv) at least oneother protein. Each protein may, or may not, be conjugated with a Histag and/or a FLAG tag, at the N and C termini, respectively. Monoclonalantibodies are commercially available for both the His and FLAG tags Adifferent unique nucleotide barcode is conjugated to a His monoclonalantibody and a FLAG monoclonal antibody. Each of the tagged proteins,are then individually incubated with the barcoded antibodies.Quantitative polymerase chain reactions (qPCR) may be used to determineyield of His/FLAG SNAPPLE junctions. Chimeric barcodes should beobtained in the presence of a protein, in proportion to the relativeproximity of the N and C termini.

Verification that the SNAPPLE biochemistry works may be performed byusing: i) EGFR conjugated with a His tag only; ii) EGFR conjugated witha FLAG tag only; and iii) EGFR conjugated with both a His tag and a FLAGtag at the N and C terminii, respectively. Highly specific antibodiesfor both His and FLAG are commercially available, wherein a first uniquenucleotide barcode is conjugated to a His monoclonal antibody (mAb), anda second unique nucleotide barcode is conjugated to a FLAG monoclonalantibody. Next, the three protein samples are individually incubatedwith each of the two barcoded monoclonal antibodies. Quantitativepolymerase chain reaction (qPCR) is then performed on each sample todetermine the yield of asymmetric junctions resulting from a ligation ofthe first barcode and the second barcode. Due to the proximity of thefirst and second barcodes in the doubly-tagged EGFR, a chimeric barcodecomprising the first and second nucleic acid barcodes are formed onlywith the double-tagged EGFR.

Verification that the SNAPPLE biochemistry can determine proximity ofmultiple targets in vitro may be performed by using: i) EGFR tagged witha His tag only; ii) Her-2 tagged with a FLAG tag only, and iii) C-Myctagged with a FLAG tag only. This combination of proteins is usefulbecause EGFR interacts with Her-2, but EGFR does not interact withc-Myc. A first unique nucleotide barcode is conjugated to a Hismonoclonal antibody, and a second unique nucleotide barcode isconjugated to a FLAG monoclonal antibody. Next, the three proteins areincubated as pairs: i) EGFR+Her-2; ii) EGFR+c-Myc; and iii) Her2+c-Myc.Quantitative polymerase chain reaction (qPCR) is then performed on eachsample to determine the yield of asymmetric junctions (i.e., forexample, an asymmetric nucleotide barcode sequence or chimeric barcode)resulting from a ligation of the first unique nucleotide barcode and thesecond unique nucleotide barcode. The asymmetric junctions comprising achimeric barcode of the first and second nucleic acid barcodes arestrongly enriched in the EGFR-Her-2 pair incubation, due to theproximity of the first and second unique nucleotide barcodes.

Verification that the SNAPPLE biochemistry can determine proximity ofmultiple targets inside a cell may be performed by using: i) EGFR taggedwith a His tag only; ii) Her-2 tagged with a FLAG tag only, and iii)C-Myc tagged with a FLAG tag only. This combination of proteins isuseful because EGFR interacts with Her-2, but EGFR does not interactwith c-Myc. A first unique nucleotide barcode is conjugated to a Hismonoclonal antibody, and a second unique nucleotide barcode isconjugated to a FLAG monoclonal antibody. Next, three cell populations(i.e., for example, U2 osteosarcoma cells) are transfected with thethree possible protein pairs: i) EGFR+Her-2; ii) EGFR+c-Myc; and iii)Her-2+c-Myc. Quantitative polymerase chain reaction (qPCR) is thenperformed on each sample to determine the yield of asymmetric junctionsresulting from a ligation of the first unique barcode and the secondunique barcode. The asymmetric junctions comprising a chimeric barcodeof the first and second unique nucleotide barcodes are strongly enrichedin the EGFR-Her-2 pair transfected cells, due to the proximity of thefirst and second unique nucleotide barcodes.

Verification that the SNAPPLE biochemistry can determine proximity ofmultiple targets in vivo may be performed by using; i) EGFR; ii) Her-2;and iii) C-Myc. A first unique nucleotide barcode is conjugated to aEGFR monoclonal antibody. A second unique nucleotide barcode isconjugated to a Her-2 monoclonal antibody. A unique third nucleotidebarcode is conjugated to a c-Myc monoclonal antibody. Next, three cellpopulations (i.e., for example, U2 osteosarcoma cells) are transfectedwith the three possible protein pairs: i) EGFR+Her-2; ii) EGFR+c-Myc;and iii) Her-2+c-Myc. Quantitative polymerase chain reaction (qPCR) isthen performed on each sample to determine the yield of asymmetricjunctions resulting from a ligation of the various barcodes. Theasymmetric junctions comprising a chimeric barcode of the first andsecond unique nucleic acid barcodes are strongly enriched in theEGFR-Her-2 pair transfected cells, due to the proximity of the first andsecond unique nucleotide barcodes.

B. Locked Nucleic Acid (LNA) Binding Partners

In one embodiment, the present invention contemplates a compositioncomprising a first single stranded forked adapter sequence, wherein theadapter sequence encodes a binding partner. In one embodiment, thebinding partner comprises a locked nucleic acid (LNA) sequence. In oneembodiment, the binding partner is attached to a unique nucleotidebarcode sequence. See, FIG. 18. Synthetic procedures for locked nucleicacids may be found. Singh et al, Chem. Comm. 455-456 (1998); and WengelJ., Acc. Chem. Res., 32:301-310 (1998).

Locked Nucleic Acid (LNA) was first described as a class ofconformationally restricted oligonucleotide analogues. The design andability of oligos containing locked nucleic acids (LNAs) to bindsupercoiled, double-stranded plasmid DNA in a sequence-specific manner.The main mechanism for LNA oligos binding plasmid DNA has beendemonstrated to be by strand displacement. LNA oligos are more stablybound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’for procedures such as particle mediated DNA delivery (gene gun). It wasshown that LNA oligos remain associated with plasmid DNA after cationiclipid-mediated transfection into mammalian cells.

LNA oligos can bind to DNA in a sequence-specific manner so that bindingdoes not interfere with plasmid conformation or gene expression. LNAoligonucleotides exhibit thermal stabilities towards complementary DNAand RNA, which allows mismatch discrimination. The high binding affinityof LNA oligos allows for the use of short probes in antisense protocolsand LNA is recommended for use in any hybridization assay that requireshigh specificity and/or reproducibility, e.g., dual labeled probes, insitu hybridization probes, molecular beacons and PCR primers.Furthermore, LNA offers the possibility to adjust T_(m) values ofprimers and probes in multiplex assays. Each LNA base addition in anoligo increases the T_(m), by approximately 8° C. As a result of thesesignificant characteristics, the use of LNA-modified oligos in antisensedrug development is now coming under investigation, and recently thetherapeutic potential of LNA has been reviewed.

The synthesis and incorporation of LNA bases can be achieved by usingstandard DNA synthesis chemistry. Detailed research results have not yetconcluded as to the amount of LNA bases and regular DNA base combinationin successful antisense and gene delivery experiments. Due to the highaffinity and thermal stability of the LNA: DNA duplex it is not advisedto have more than 15 LNA bases in an oligo; this induces strongself-hybridization.

The use of LNA C base requires special synthesis and post synthesisprotocols. LNA-containing oligonucleotides can be purified and analyzedusing the same methods employed for standard DNA. LNA can be mixed withDNA and RNA, as well as other nucleic acid analogues, modifiers andlabels. LNA oligonucleotides are water soluble, and can be separated bygel electrophoresis and precipitated by ethanol.

Specific types of locked-nucleic Acid (LNA) phosphoramidites have beenreported. U.S. Pat. No. 6,268,490 (herein incorporated by reference).Other embodiments are illustrated herein. See, FIG. 20.

An LNA monomer can refer to a conformationally restricted nucleotideanalogue with an extra 2′-O, 4′-C-methylene bridge added to the ribosering that may exhibit enhanced hybridization affinity towardscomplementary DNA and RNA. Evaluations of the influence of LNA residueson hybridization thermodynamics, counterions and hydration of DNA-DNAand DNA-RNA heteroduplexes were performed using spectroscopic andcalorimetric techniques. Thermodynamic analysis for duplex formationusing UV and differential scanning calorimetry suggested thatLNA-induced stabilization results from a large, favorable increase inthe enthalpy of hybridization that compensates for the unfavorableentropy change. The heat capacity change (ΔC_(p)) accompanying theduplex formation may obtained through differential scanning calorimetry(DSC). Furthermore, it was observed that relative to the formation ofunmodified duplex, the formation of LNA-modified duplexes may beaccompanied by a higher uptake of counterions and a lower uptake ofwater molecules. Kaur et al., “Thermodynamic, counterion and hydrationeffects for the incorporation of locked nucleic acid (LNA) nucleotidesin duplex” Nucleic Acids Symp Ser (Oxf). 52:425-426 (2008).

Ligation-based methods have been disclosed for identifying at least twotarget nucleotides in a mixed population sample, that is a sample thatcontains or potentially contains target nucleic acid sequences from morethan one source. Typically, two ligation reaction compositionscomprising locked nucleic acids are formed, ligation products generated,and the ligation products or their surrogates are analyzed to identifytarget nucleotides in the mixed population sample. In certainembodiments, the target nucleic acid sequences, the ligation products,or both are amplified. In certain embodiments, multiplex amplificationand/or ligation reactions are performed. Karger et al., “Methods andkits for identifying target nucleotides in mixed populations” U.S. Pat.No. 7,427,479 (herein incorporated by reference).

In one embodiment, the present invention contemplates a SNAPPLE probecomprising an LNA binding partner may have affinity for an RNA. Forexample, an RNA may comprise a messenger RNA (mRNA). In one embodiment,the mRNA binds a plurality of different SNAPPLE probes at differentbinding sites. Although it is not necessary to understand the mechanismof an invention, it is believed that two SNAPPLE probes may bind todifferent regions of an RNA molecule, demonstrating that these regionscome into close proximity. See, FIG. 19.

In one embodiment, the present invention contemplates a SNAPPLE probecomprising an LNA binding partner may have affinity for a DNA. Forexample, a DNA may comprise a double stranded DNA, a single strandedDNA, or a cDNA. In one embodiment, the DNA binds a plurality ofdifferent SNAPPLE probes at different binding sites. Although it is notnecessary to understand the mechanism of an invention, it is believedthat two SNAPPLE probes may bind to different regions of an DNAmolecule, demonstrating that these regions come into close proximity.

C. Intracellular Receptor Binding Partners

Two basic types of receptor transducing systems have been reported: i)those which utilize transmembrane receptors that may be activated, forexample, at the cell surface by an appropriate hormone or biochemical,thereby releasing intracellular second messenger molecules (i.e. cAMP),and; ii) those that utilize internal, cytoplasmic or nuclear receptors(i.e., for example, intracellular receptors) which, upon activation, mayinteract directly with DNA to alter the genetic program of a cell, orfacilitate other intracellular biochemical processes (i.e., for example,calcium regulation). McDonnell et al., “Nuclear hormone receptors astargets for new drug discovery” Biotechnology (NY) 11:1256-1261 (1993).

In one embodiment, the present invention contemplates a SNAPPLE probecomprising a binding partner, wherein the partner comprises anintracellular receptor molecule. In one embodiment, the receptormolecule may comprise a membrane bound receptor. In one embodiment, thereceptor molecule may comprise a soluble receptor. In one embodiment,the intracellular receptor may have affinity for a binding ligandselected from the group comprising a peptide, a hormone, aneurotransmitter, or cofactors.

In one embodiment, the present invention contemplates a method providingat least two SNAPPLE probes, wherein each probe comprises affinity for adifferent binding ligand for detecting the spatial proximity between atleast two different intracellular receptor molecules.

In one embodiment, the present invention contemplates a method providingat least two SNAPPLE probes, wherein each probe comprises a receptorsubunit as a binding partner. In one embodiment, the first probecomprises a first receptor subunit. In one embodiment, the second probecomprises a second receptor subunit. In one embodiment, the secondbinding ligand comprises an affinity for a cofactor subunit. Although itis not necessary to understand the mechanism of an invention, it isbelieved that binding the probes to a receptor may determineconformation changes in a receptor molecule upon interaction with abinding ligand.

1. Calcium Regulation

It is believed that an intracellular receptor specific for cyclicADP-ribose (cADPR) exists and is different from the inositoltrisphosphate (IP3) receptor. cADPR is a metabolite of NAD⁺ which is asactive as IP3 in mobilizing intracellular Ca²⁺ in sea urchin eggs. Theenzyme responsible for synthesizing cADPR is found not only in seaurchin eggs but also in various mammalian tissue extracts, suggestingthat it may be a general messenger for Ca²⁺ mobilization in cells. Seaurchin eggs were homogenized and the Ca²⁺-storing microsomes wereseparated from mitochondria and other organelles by Percoll densitycentrifugation. Radioactive cADPR with high specific activity wasproduced by incubating [³²P]NAD⁺ with the synthesizing enzyme and theproduct purified by high pressure liquid chromatography. The enzyme wasmembrane bound and was isolated from dog brain extracts by sucrosedensity gradient centrifugation. Partial purification of the enzyme wasachieved by DEAE ion-exchange chromatography after solubilization with3-[(cholamidopropyl) dimethylammonio]-1-propanesulfonate. Specificbinding of ³²P-labeled cADPR to a saturable site on the Ca²⁺-storingmicrosomes was detected by a filtration assay. Scatchard analysisindicated a binding affinity of about 17 nM and a capacity of about 25fmol/mg protein. The binding was not affected by either NAD⁺ (theprecursor) or ADP-ribose (the hydrolysis product) at 0.5 μM but waseliminated by 0.3 μM nonlabeled cADPR. The receptor for cADPR appearedto be different from that of IP3 since IP3 was not an effectivecompetitor at a concentration as high as 3 μM. Similarly, heparin at aconcentration that inhibits most of the IP3-induced calcium release fromthe microsomes did not affect the binding. The binding showed aprominent pH optimum at about 6.7. Calcium at 40 μM decreased thebinding by about 50%. These dependencies of the binding on pH and Ca²⁺are different from those reported for the IP3 receptor and providefurther support that the intracellular receptors for cADPR and IP3 aredifferent. Lee H C., “Specific binding of cyclic ADP-ribose tocalcium-storing microsomes from sea urchin eggs” J Biol Chem.266:2276-2281 (1991).

Ca²⁺ mobilization is believed mediated by intracellular receptors havingaffinity for inositol 1,4,5-trisphosphate (IP3). IP3 is believed to be asecond messenger generated via receptor-stimulated hydrolysis ofphosphatidylinositol 4,5-bisphosphate. Various reports suggest IP3intracellular receptor localization at various subcellular structuresincluding, but not limited to: i) elements of the endoplasmic reticulum(both rough and smooth surfaced regions); ii) the nuclear envelope, andiii) the plasma membrane. Immunofluorescent polyclonal monospecificantibodies directed against the inositol 1,4,5-trisphosphate receptor incentral nervous system tissue detected receptors localized in Purkinjecells, whereas the cerebellar cortex remained negative. The visualizedIP3 receptors were concentrated in cisternal stacks (piles of up to 12parallel cisternae separated by regularly spaced bridges, located bothin the deep cytoplasm and beneath the plasma membrane; average density,greater than 5 particles/micron of membrane profile); in cisternalsinglets and doublets adjacent to the plasma membrane (average density,approximately 2.5 particles/micron); and in other apparentlysmooth-surfaced vesicular and tubular profiles. In the dendrites,approximately half of the nonmitochondrial, membrane-bound structures(cisternae, tubules, and vesicles), as well as small cisternal stacks,were labeled. Dendritic spines always contained immunolabeled cisternaeand vesicles. These results identify a large, smooth-surfacedendoplasmic reticular subcompartment that appears to play a role in thecontrol of Ca²⁺ homeostasis. Satoh et al., “The inositol1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitativeimmunogold labeling reveals concentration in an ER subcompartment” JCell Biol. 111:615-624 (1990).

The visinin-like protein (VSNL) subfamily, including VILIP-1 (thefounder protein), VILIP-2, VILIP-3, hippocalcin, and neurocalcin delta,constitute a highly homologous subfamily of intracellular neuronalcalcium sensor (NCS) proteins. These proteins display differences intheir calcium affinities, in their membrane-binding kinetics, and in theintracellular targets to which they associate after calcium binding.Even though the proteins use a similar calcium-myristoyl switchmechanism to translocate to cellular membranes, they showcalcium-dependent localization to various subcellular compartments whenexpressed in the same neuron. These distinct calcium-myristoyl switchproperties might be explained by specificity for defined phospholipidsand membrane-bound targets; this enables VSNLs to modulate variouscellular signal transduction pathways, including cyclic nucleotide andMAPK signaling. VSNLs may directly or indirectly effect gene expressionand/or interact with components of membrane trafficking complexes,thereby having a possible role in membrane trafficking of differentreceptors and ion channels, including, but not limited to, glutamatereceptors of the kainate and AMPA subtype, nicotinic acetylcholinereceptors, and Ca²⁺-channels. Braunewell et al., “Visinin-like proteins(VSNLs): interaction partners and emerging functions in signaltransduction of a subfamily of neuronal Ca²⁺-sensor proteins” CellTissue Res. 335(2):301-316 (2009).

2. Eicosanoids

Eicosanoids are produced by many different cell types through theirligation and activation of specific membrane-bound and intracellularreceptors. They are believed to regulate a myriad of physiological andpathological functions, including, for example, body temperature. Whilethe thermoregulatory role of eicosanoids has mainly been associated withillness-induced fever, they are unlikely to be involved in themaintenance of normal body temperature. Aronoff et al., “Eicosanoids innon-febrile thermoregulation” Prog Brain Res 162:15-25 (2007).

3. Protein Kinase C

Protein kinase C (PKC) translocates from the soluble to the cellparticulate fraction on activation. Intracellular receptors that bindactivated PKC in the particulate fraction have been implicated by anumber of studies. Previous work identified 30- to 36-kDa proteins inthe particulate fraction of heart and brain that bound activated PKC ina specific and saturable manner. These proteins were termedintracellular Receptors for Activated C-Kinase, or RACKs. Cloning of acDNA encodes a 36-kDa protein (RACK1) that comprises RACK functionality.for example: (i) RACK1 bound PKC in the presence of PKC activators, butnot in their absence; (ii) PKC binding to the recombinant RACK1 was notinhibited by a pseudosubstrate peptide or by a substrate peptide derivedfrom the pseudosubstrate sequence, indicating that the binding did notreflect simply PKC association with its substrate; (iii) binding of PKCto RACK1 was saturable and specific; two other protein kinases did notbind to RACK1; (iv) RACK1 contains two short sequences homologous to aPKC binding sequence previously identified in annexin I and in the brainPKC inhibitor KCIP. Further, peptides derived from these sequencesinhibited PKC binding to RACK1. In vitro data also suggest a role forRACK1 in PKC-mediated signaling. Ron et al., “Cloning of anintracellular receptor for protein kinase C: a homolog of the betasubunit of G proteins” Proc Natl Acad Sci USA. 91:839-843 (1994). RACK1is a homolog of the beta subunit of G proteins, which were recentlyimplicated in membrane anchorage of the beta-adrenergic receptor kinase.Pitcher et al., Science 257:1264-1267 (1992).

Isoforms of the phospholipid-dependent protein kinase, protein kinase C(PKC), may also be intracellular receptors for diacylglycerol.Cytoplasmic nPKCΔ and nPKCε have been reported to detect increases inmembrane diacylglycerols and translocate to the membrane. This bringsabout PKC activation, though modifications additional to binding tophospholipids and diacylglycerol are involved. The next event (probablyassociated with PKC activation) is the activation of the membrane-boundsmall G protein Ras by exchange of GTP for GDP. RasGTP loadingtranslocates Raf family mitogen-activated protein kinase (MAPK) kinasekinases to the membrane, initiates the activation of Raf, and thusactivates the extracellular signal-regulated kinase ½ (ERK½) cascade.Over longer times, two analogous protein kinase cascades, the c-JunN-terminal kinase and p38-mitogen-activated protein kinase cascades,become activated. As the signals originating from the ET(A) receptor aretransmitted through these protein kinase pathways, other signalingmolecules become phosphorylated, thus changing their biologicalactivities. For example, ET-1 increases the expression of the c-juntranscription factor gene, and increases abundance and phosphorylationof c-Jun protein. These changes in c-Jun expression and phosphorylationare likely to be important in the regulation of gene transcription.Sugden et al., “Endothelin signalling in the cardiac myocyte and itspathophysiological relevance” Curr Vase Pharmacol. 3:343-351 (2005).

4. Steroids

Interactions between polymers and intracellular estrogen receptors werestudied in the context of tumoral indicators of breast cancer. Thesepolymers were used as microcarriers for MCF7 cell cultures, a cellularmodel of human breast cancer. Quantification of MCF7 cell estrogenreceptors was determined by radioligand binding assay for different daysof cellular proliferation. These polymers demonstrated an inverserelationship between inhibition of cell proliferation and increasedintracellular estrogen receptors. Mestries et al., “Interactions betweenbiospecific polymers and MCF7 cells: modulation of cellularproliferation and expression of estrogen receptors” Bull Cancer.84:1017-1023 (1997)

In the past, the intracellular response of target cells to the steroidhormone aldosterone has been divided into acute nongenomic (<10 min) andsustained genomic (>10 min) action. Atomic force microscopy (AFM)observations in living endothelial cells demonstrate that aldosteroneinduces cell volume increase in less than 10 minutes, therebyidentifying the cell nucleus as the swelling site. Hormone-inducednuclear swelling can reach 15 to 28% of total cell volume dissipatingwithin 30 minutes. AFM-investigation of the intracellular signal pathwayin nuclear envelope of aldosterone-injected Xenopus laevis oocytesvisualizes putative intracellular receptors (40 kD granules) bound tonuclear pores 2 minutes after hormone injection, with subsequentmacromolecule translocation into the nucleus. 15 minutes latermacromolecules (800 kD plugs) appear in the central channels of thenuclear pores. The plugs resemble ribonucleoproteins that carry thealdosterone-induced mRNA to the ribosomes. It is believed thatsteroid-induced nuclear swelling is caused by a shift ofreceptors/transcription factors from cytoplasm into nucleoplasm followedby gene transcription. Nuclear volume returns to normal when mRNA exportthrough the nuclear pores is finished. Thus, steroid-inducednet-movements of macromolecules between intracellular compartmentsinitiate shifts in cell volume compensated by volume regulatorytransporters and ion channels in the plasma membrane. Oberleithner etal., “Aldosterone and nuclear volume cycling” Cell Physiol Biochem10:429-434 (2000).

It is believed that intracellular corticosteroid receptors may mediatetissue effects of glucocorticoids in vertebrates including, but notlimited to, two intracellular receptors that act primarily asligand-activated transcription factors and a membrane-associatedreceptor. Some intracellular steroid receptor subtypes have been wellcharacterized in mammals. Breuner et al., “Pharmacologicalcharacterization of intracellular, membrane, and plasma binding sitesfor corticosterone in house sparrows” Gen Comp Endocrinol. (Epub. Feb.21, 2009)

Many of the biological actions of progestins may depend on binding tointracellular receptors and through a long chain of events to subsequentstimulation of transcriptional activity and protein synthesis. Thisprocess requires at least a few hours in time and many differentcoregulator proteins play a role after progestin binding to itsreceptor. Thijssen J H., “Gene polymorphisms that may influence thebiological effects of progestins” Maturitas (Epub Jan. 6, 2009).

5. Apoptosis

Ribonucleic acid interference (RNAi)-mediated knockdown of theintracellular receptors NALP3 or MDA5 did not affect poly(I:C)-inducedpro-IL-1beta mediated apoptosis. Stimulation of membrane-bound Toll-likereceptors (TLRs) may up-regulate pro-IL-1β expression, activatecaspase-1, and is believed to be mainly initiated by cytosolic Nod-likereceptors. Polyinosinic:polycytidylic acid (poly[I:C]) andlipopolysaccharide stimulation of macrophages may induce pro-IL-1βprocessing via a Toll/IL-1R domain-containing adaptor-inducinginterferon-beta-dependent signaling pathway that is initiated by TLR3and TLR4, respectively. Consequently, caspase-8 may play a role in theproduction of biologically active IL-1β in response to TLR3 and TLR4stimulation. Maelfait et al., “Stimulation of Toll-like receptor 3 and 4induces interleukin-1β maturation by caspase-8” J Exp Med. 205:1967-1973(2008).

Toxic agents, particularly those that exert their actions with a greatdeal of specificity, sometimes act via intracellular receptors to whichthey bind with high affinity. Some examples include, but are not limitedto, soluble intracellular receptors, which may be important in mediatingtoxic responses. For example, an intracellular glucocorticoid receptormay mediate toxicity associated effects such as apoptosis of lymphocytesas well as neuronal degeneration as a response to stress. The peroxisomeproliferator activated receptor (PPAR) may be associated withhepatocarcinogenesis in rodents. The dioxin receptor may mediate a moregeneralized response to toxin exposure. Gustafsson J A.,“Receptor-mediated toxicity” Toxicol Lett. 82-83:465-470 (1995)

6. Hormone Regulation

In HEK 293 and COS7 cells, thyrotropin-releasing hormone (TRH) receptorsare believed to be predominantly intracellular. In transientlytransfected COS7 cells, the TRH receptor colocalized with endoplasmicreticulum and Golgi markers. The pattern of TRH receptorimmunofluorescence was the same over a wide range of receptor expressionin transiently transfected COS7 cells, and all cell lines bound similaramounts of ³H- and rhodamine-labeled TRH analogs, suggesting thatcell-specific differences in TRH receptor localization were not simplythe result of overexpression. In all cell contexts, TRH receptors on theplasma membrane underwent extensive ligand-driven endocytosis.Inhibitors of glycosylation did not alter the subcellular distributionof receptors. In HEK 293 cells expressing the transfected TRH receptor,protein synthesis inhibitors caused translocation of intracellularreceptors to the cell surface, as shown by a marked increase in cellsurface immunofluorescence and [³H][N3-methyl-His2]TRH binding. Thelocalization of an epitope-tagged receptor for TRH expressed indifferent cell contexts was studied with immunofluorescence microscopy.In pituitary lactotrophs, which normally express TRH receptors, and inAtT20 pituitary corticotrophs, TRH receptor immunoreactivity wasprimarily confined to the plasma membrane. These results demonstratethat the subcellular localization of the TRH receptor depends on thecell context in which it is expressed and that intracellular receptorsare capable of translocation to the plasma membrane. Yu et al., “Effectof cell type on the subcellular localization of thethyrotropin-releasing hormone receptor” Mol Pharmacol. 51:785-793(1997).

7. Nitric Oxide

It has been suggested that, in physiological conditions, myoglobin actsas intracellular scavenger preventing nitric oxide (NO) from reachingits intracellular receptors in cardiomyocytes. In myoglobin-deficientconditions, NO is able to reduce contractility via activation of thesoluble guanylyl cyclase/cyclic GMP pathway. NO donors may include, butare not limited to, S-nitroso-N-acetylpenicillamine (SNAP),sodium(Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (DEA-NONOate),and (Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate). Specifically, SNAPslightly reduced contractility in preparations from wild type mice atconcentrations above 100 μM and was more pronounced in myo^(−/−) mice.DEA-NONOate and DETA-NONOate also reduced contractility in preparationsfrom myo^(−/−) mice more than wild type mice. Pre-incubation with aninhibitor of the soluble guanylyl cyclase (i.e., for example,1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 100 μM) prevented theeffects of the NO donors on contractility in myo^(−/−) mice. Wegener etal., “Effects of nitric oxide donors on cardiac contractility inwild-type and myoglobin-deficient mice” Br J Pharmacol. 136:415-420(2002).

D. Biological Cell Binding Partners

In one embodiment, the present invention contemplates a SNAPPLE probecomprsing a binding partner, wherein the partner comprising a biologicalcell. In one embodiment, the cell comprising a blood cell. In oneembodiment, the blood cell comprises a white blood cell. In oneembodiment, the white blood cell comprises a lymphocyte. In oneembodiment, the cell comprises and antigen presenting cell. In oneembodiment, the cell comprises a stem cell. In one embodiment, the stemcell comprises a bone marrow cell. In one embodiment, the stem cellcomprises an embryonic stem cell. In one embodiment, the stem cellcomprises an epithelial stem cell. In one embodiment, the stem cellcomprises an endothelial stem cell. In one embodiment, the endothelialstem cell comprises a cardiovascular endothelial stem cell.

E. Derivatized Bead Binding Partners

In one embodiment, the present invention contemplates a SNAPPLE probecomprsing a binding partner, wherein the partner comprising aderivatized bead. In one embodiment, the bead comprises an Fab fragment.In one embodiment, the bead comprises an LNA sequence. In oneembodiment, the bead comprises a DNA sequence. In one embodiment, thebead comprises a small organic molecule. In one embodiment, the smallorganic molecule comprises a drug.

A preferred embodiment includes polystyrene beads, between 10-100microns in diameter, which are capable of substantially homogeneousdispersion and separation from a medium by filtration or floatation.Another preferred embodiment includes ferromagnetic beads. Aferromagnetic bead marketed under the trademarks BIO-MAG is capable ofsubstantially homogeneous dispersion in an aqueous medium and can beretrieved or immobilized by an electromagnetic field. The ferromagneticbead includes an iron core which is coated with an amine reactivecovering. The beads are generally spherical and have a diameter of onemicron. The polystyrene and ferromagnetic beads are treated to includeantiligand moieties.

Beads (i.e., for example, a polystyrene bead) having reactive aminefunctional groups can be reacted with polynucleotides to covalentlyaffix the polynucleotide to the bead. The beads are reacted with 10percent glutaraldehyde in sodium phosphate buffer and subsequentlyreacted in a phosphate buffer with ethylene-diamine adduct of thephosphorylated polynucleotide.

One method of covalently binding a binding partner to derivatize a beadcomprises a chemical conjugation agent to activate the bead, followed bythe attachment of the binding partner to the conjugation agent.Synthetic polymeric beads are advantageous because they can withstandharsh derivatization conditions and are relatively inexpensive, andoften yield a linkage that is stable to a wide range of denaturingconditions. A number of derivatized beads are commercially available,all with various constituents and sizes. Beads formed from syntheticpolymers include, but are not limited to, polyacrylamide, polyacrylate,polystyrene, or latex are commercially available from numerous sourcessuch as Bio-Rad Laboratories (Richmond, Calif.) and LKB Produkter(Stockholm, Sweden). Beads formed from natural macromolecules andparticles include, but are not limited to, agarose, crosslinked agarose,globulin, deoxyribose nucleic acid, and liposomes are commerciallyavailable from sources such as Bio-Rad Laboratories, Pharmacia(Piscataway, N.J.), and IBF (France). Beads formed from copolymers ofpolyacrylamide and agarose are commercially available from sources suchas IBF and Pharmacia. Magnetic beads are commercially available fromsources such as Dynal Inc. (Great Neck, N.Y.).

F. Small Organic Molecule Binding Partners

In one embodiment, the present invention contemplates a SNAPPLE probecomprising a binding partner, wherein the partner comprises a smallorganic molecule. In one embodiment, the small organic molecule maycomprise a second messenger. In one embodiment, the small organicmolecule may comprise an enzymatic cofactor. In one embodiment, thesecond messenger may include, but not limited to, cyclic AMP, cyclicGMP, prostaglandins, diacylglycerols, or an inositol phosphate.

III. Asymmetric Barcode Amplification

In one embodiment, the present invention contemplates a compositioncomprising an asymmetric nucleotide barcode sequence. In one embodiment,the present invention contemplates a method comprising amplifying anasymmetric nucleotide barcode thereby creating a plurality of amplicons.In one embodiment, the amplicon comprises the nucleic acid sequence ofthe asymmetric nucleotide barcode. In one embodiment, the method furthercomprises identifying the amplicon nucleic acid sequence by a sequencingtechnique. In one embodiment, the method further comprises comparing theamplicon nucleic acid sequence to a SNAPPLE probe unique nucleotidebarcode. In one embodiment, the method further comprises identifying anintracellular component associated with the SNAPPLE probe uniquenucleotide barcode.

Polymerase chain reaction (PCR) technology amplifies a specific DNAsegment when flanked by a set of primers. R. K. Saiki et al., Science230, 1350 (1985); Erlich, D. Gelfand, J. J. Sninsky, Science 252, 1643(1991). The PCR process thereby allows the production of large amountsof specific DNA products (i.e., for example, amplicons), which can bedetected by various methods (i.e., for example, gel electrophoresisisolation and sequencing). The selected primer pair is believedresponsible for the high specificity of PCR for a target sequence. PCRwas initially used to detect antigen-antibody complexes in a protocoltermed, Immuno-PCR. In Immuno-PCR, a linker molecule with bispecificbinding affinity for DNA and antibodies is used to attach a DNA molecule(marker) specifically to an antigen-antibody complex, resulting in theformation of a specific antigen-antibody-DNA conjugate. The presentinvention improves upon the basic tenets of Immuno-PCR such that morethan one target antigen can be identified, in addition to providingspatial proximity information.

IV. Intracellular Component Contact Maps

In one embodiment, the present invention contemplates a method,providing a plurality of SNAPPLE probes and introducing the probes intoa cell, wherein the cell comprises a plurality of intracellularcomponents. In one embodiment, at least one probe comprises a bindingpartner having affinity for a first intracellular component. In oneembodiment, the intracellular component comprises a protein. In oneembodiment, the intracellular component comprises an intracellularreceptor. In one embodiment, the intracellular component comprises asmall organic molecule. In one embodiment, the cell comprises a cellcycle phase. In one embodiment, the cell is exposed to a specifictemperature. In one embodiment, the cell is exposed to a drug. In oneembodiment, the cell is exposed to a toxin. In one embodiment, the cellis exposed to radiation. In one embodiment, the method further comprisesbinding the binding partner to the intracellular component underconditions such that the probe pairs create an asymmetric nucleotidebarcode. In one embodiment, the method further comprises identifying theasymmetric nucleotide barcodes, thereby creating an intracellularcomponent contact map. In one embodiment, the contact map comprises aheat map.

Color mapping of intracellular component paring data using contour colormapping approaches may be found in two, three, and four dimensionalcontour heatmaps. Contour color heatmapping uses the entire data spaceor data matrix (image) as the basis for the color process. Colorintensity may thereby reflect that amount of data being processed forany particular data point (i.e, is proportional to the frequency ofisolated asymmetric nucleotide barcode sequences).

In one embodiment, the present invention contemplates a heatmapcomprising an array comprising rows and columns. In one embodiment, therows comprise UNB's intracellular compnents A-Z. In one embodiment, thecolumns comprise DNB's for intracellular components A-Z. In oneembodiment, an array comprising a color intensity at a specificrow-column intersection indicates that the row intracellular componentand the column intracellular component interact (i.e., were present inclose proximity). In one embodiment, the color intensity is proportionalto the observed frequency of the intracellular component interaction.

Systems biology aims to understand biological systems on a comprehensivescale, such that the components that make up the whole are connected toone another and work through dependent interactions. Molecularcorrelations and comparative studies of molecular expression canestablish interdependent connections in systems biology. Commerciallyavailable software packages provide limited data mining capability.These programs require the user to first generate visualization datawith a preferred data mining algorithm and then upload the resultingdata into the visualization package for graphic visualization ofmolecular relations. Alternative interactive visual data miningapplications, (i.e., for example, SysNet) provide an interactiveenvironment for the analysis of high data volume molecular expressioninformation of most any type from biological systems. The interactivenature of the program presents intermolecular correlation informationcompatible with heatmap layouts. Zhang et al., “Interactive analysis ofsystems biology molecular expression data” BMC Syst Biol. 2:23 (2008).

Large quantities of chemical structure and biological activity databrought about through combinatorial chemistry and high-throughputscreening technologies has created the need for sophisticated graphicaltools to evaluate the data. Many chemoinformatics software applicationsapply standard clustering techniques to organize structure-activitydata, but they differ significantly in their approaches to visualizingclustered data. For example, Molecular Property eXplorer (MPX) canpresents clustered data in the form of heatmaps. MPX employsagglomerative hierarchical clustering to organize data on the basis ofthe similarity between 2D chemical structures or similarity across apredefined profile of biological assay values. Visualization ofhierarchical clusters as heatmaps provides simultaneous representationof cluster members along with their associated assay values. Heatmapsprovide visualization of the cluster members across an activity profile.Kibbey et al., “Molecular Property eXplorer: a novel approach tovisualizing SAR using tree-maps and heatmaps” J Chem Inf Model.45:523-32 (2005).

A. Cell Cycles

The cell cycle, or cell-division cycle, is the series of events thattake place in a cell leading to its division and duplication(replication). In cells without a nucleus (prokaryotes), the cell cycleoccurs via a process termed binary fission. In cells with a nucleus(eukaryotes), the cell cycle can be divided in two brief periods:interphase—during which the cell grows, accumulating nutrients neededfor mitosis and duplicating its DNA—and the mitotis (M) phase, duringwhich the cell splits itself into two distinct cells, often called“daughter cells”. The cell-division cycle is a vital process by which asingle-celled fertilized egg develops into a mature organism, as well asthe process by which hair, skin, blood cells, and some internal organsare renewed. The cell cycle consists of four distinct phases: G₁ phase,S phase (synthesis), G₂ phase (collectively known as interphase) and Mphase (mitosis). M phase is itself composed of two tightly coupledprocesses: mitosis, in which the cell's chromosomes are divided betweenthe two daughter cells, and cytokinesis, in which the cell's cytoplasmdivides forming distinct cells. Activation of each phase is dependent onthe proper progression and completion of the previous one. Cells thathave temporarily or reversibly stopped dividing are said to have entereda state of quiescence called G₀ phase.

Prokaryotic cell cycles have been studied using bacterial models.Despite their small size and lack of obvious intracellular structures,bacteria have a complex and dynamic intracellular organization. Recentwork has shown that many proteins, and even regions of the chromosome,are localized to specific subcellular regions that can change over time,sometimes extraordinarily fast. Protein function can depend on cellularposition, so the analysis of the intracellular location of a protein canbe crucial for understanding its activity. Because regulatory proteinsare among those that reside at specific cellular sites, it is nownecessary to consider three-dimensional organization when describing thegenetic networks that control bacterial cells. Jensen et al., “Proteinson the move: dynamic protein localization in prokaryotes” Trends CellBiol. 10:483-488 (2000).

Bacteria exhibit a high degree of intracellular organization, both inthe timing of essential processes and in the placement of thechromosome, the division site, and individual structural and regulatoryproteins. Mechanisms that control timing of cell cycle and developmentalevents include transcriptional cascades, regulated phosphorylation andproteolysis of signal transduction proteins, transient geneticasymmetry, and intercellular communication. Surprisingly, many signaltransduction proteins are dynamically localized to specific subcellularaddresses during the cell division cycle and sporulation, and properlocalization is essential for their function. For example, the Minproteins that govern division site selection in Escherichia coli may bethe first example of a system that generates positional information denovo. Ryan et al., “Temporal and spatial regulation in prokaryotic cellcycle progression and development” Annu Rev Biochem. 72:367-394 (2003).

The small nuclear GTPase Ran is believed to control the directionalityof macromolecular transport between the bacterial nucleus and thecytoplasm. Ran also may have a role during mitosis, when the nucleus isreorganized to allow chromosome segregation. Therefore, Ran may directthe assembly of the mitotic spindle, nuclear-envelope dynamics and thetiming of cell-cycle transitions. Such functions reflect the spatial andtemporal coordination of the changes that occur in intracellularorganization during the cell-division cycle. Clarke et al., “Spatial andtemporal coordination of mitosis by Ran GTPase” Nat Rev Mol Cell Biol.9:464-477 (2008).

A-kinase-anchoring proteins (AKAP) may help regulate the intracellularorganization of cyclic AMP-dependent kinase (PKA) and actin withinsomatic cells. Elevated levels of cAMP also help maintain meiotic arrestin immature oocytes, with AKAPs implicated as potential mediators.Studies have suggested that WAVE1 sequestration to the nucleus may occurduring fertilization, and is an actin-independent event that relies ondynamic microtubules but not nuclear pores. Rawe et al., “WAVE1intranuclear trafficking is essential for genomic and cytoskeletaldynamics during fertilization: cell-cycle-dependent shuttling betweenM-phase and interphase nuclei.” Dev Biol. 276:253-267 (2004)

The high mobility group N (HMGN) proteins are a family of nuclearproteins that bind to nucleosomes, changes the architecture ofchromatin, and enhances transcription and replication from chromatintemplates. The intracellular organization of the HMGN (previously knownas HMG-14/17) proteins is dynamic and is related to both cell-cycle andtranscriptional events. These proteins roam the nucleus, perhaps as partof multiprotein complexes, and their target interactions are modulatedby posttranslational modifications. Functional studies on HMGN proteinsprovide insights into the molecular mechanisms by which structuralproteins affect DNA-dependent activities in the context of chromatin.Bustin M., “Chromatin unfolding and activation by HMGN chromosomalproteins” Trends Biochem Sci. 26:431-437 (2001)

B. Temperature

It has been previously demonstrated that plant cells (i.e., for example,Nicotiana plumbaginifolia) react to cold-shock by an immediate rise incytosolic calcium. Cytoskeleton organization has also been shown to haveprofound influences on the temperature-induced calcium response. Forexample, the disruption of the microtubule meshwork by various activedrugs, such as colchicin, oryzalin and vinblastin, leads to increases inthe cytosolic calcium (up to 400 nM) in cold-shocked protoplasts overcontrol. The cytoskeleton may play a role in controlling the intensityof calcium responses to an extracellular stimulus (i.e., for example,temperature fluctuations). Mazars et al., “Organization of cytoskeletoncontrols the changes in cytosolic calcium of cold-shocked Nicotianaplumbaginifolia protoplasts” Cell Calcium 22:413-20 (1997).

C. Drugs And Toxins

The transition of adult rat aortic smooth muscle cells from acontractile to a synthetic phenotype during the first week of primaryculture on a substrate of fibronectin in serum-free medium was studiedby light and electron microscopy. The weak base chloroquine and thecarboxylic ionophore monensin were both found to inhibit the spreadingof the cells and the accompanying changes in cellular fine structure.The exchange of myofilament bundles for a prominent rough endoplasmicreticulum and Golgi complex was delayed and vacuoles filled withincompletely degraded material accumulated in the cytoplasm. Themicrotubule-disruptive drugs colchicine and nocodazole likewise opposedthe spreading and fine structural reorganization of the cells. Mosttypically, the Golgi stacks were small and widely dispersed. Inaddition, vacuoles of the type mentioned above increased in number. Onthe other hand, there was surprisingly little effect of cytochalasin B,a drug that is supposed to interfere with the assembly of actinfilaments. The observations suggest that the phenotypic modulation ofarterial smooth muscle cells is dependent on: (a) lysosomal degradationof discarded cellular constituents, (b) active vesicular transport alongthe exocytic pathway to provide the expanding cell surface with newmembrane, and (c) a normal microtubular cytoskeleton to ensure theestablishment of a new and functionally efficient intracellularorganization. Thyberg et al., “Phenotype modulation in primary culturesof rat aortic smooth muscle cells. Effects of drugs that interfere withthe functions of the vacuolar system and the cytoskeleton” Virchows ArchB Cell Pathol Incl Mol Pathol. 59:1-10 (1990)

V. Antibodies

The present invention provides isolated antibodies (i.e., for example,polyclonal or monoclonal). In one embodiment, the present inventionprovides monoclonal antibodies that specifically bind to anintracellular component including, but not limited to, a protein, aglycoprotein, a lipid, a glycolipid, or a nucleic acid.

For example, an antibody against a protein may be any monoclonal orpolyclonal antibody, as long as it can recognize the protein. Antibodiescan be produced by using a protein of the present invention as theantigen according to a conventional antibody or antiserum preparationprocess. Any suitable method may be used to generate the antibodies usedin the methods and compositions of the present invention. For example, amonoclonal antibody may be prepared by administering a protein,optionally with a suitable carrier or diluent, to an animal (e.g., amammal) under conditions that permit the production of antibodies (i.e.,for example, immunization). For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 2 times to about 10 times. Animals suitablefor use in such methods include, but are not limited to, primates,rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animal(e.g., a mouse) whose antibody titer has been confirmed is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a tumor antigen or autoantibody of the presentinvention). For example, where a supernatant of the hybridoma is addedto a solid phase (e.g., microplate) to which antibody is adsorbeddirectly or together with a carrier and then an anti-immunoglobulinantibody (if mouse cells are used in cell fusion, anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum. Separation andpurification of a monoclonal antibody (e.g., against a cancer marker ofthe present invention) can be carried out according to the same manneras those of conventional polyclonal antibodies such as separation andpurification of immunoglobulins, for example, salting-out, alcoholicprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwherein only an antibody is collected with an active adsorbent such asan antigen-binding solid phase, Protein A or Protein G and dissociatingthe binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies from ananimal or human. For example, a complex of an immunogen (an antigenagainst the protein) and a carrier protein is prepared and an animal orhuman is immunized by the complex according to the same manner as thatdescribed with respect to the above monoclonal antibody preparation. Amaterial containing the antibody against is recovered from the immunizedanimal or human and the antibody is separated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten. In addition, various condensing agents can be used for couplingof a hapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times. The polyclonalantibody is recovered from blood, ascites and the like, of an animalimmunized by the above method. The antibody titer in the antiserum canbe measured according to the same manner as that described above withrespect to the supernatant of the hybridoma culture. Separation andpurification of the antibody can be carried out according to the sameseparation and purification method of immunoglobulin as that describedwith respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a protein expressed resultingfrom a virus infection (further including a gene having a nucleotidesequence partly altered) can be used as the immunogen. Further,fragments of the protein may be used. Fragments may be obtained by anymethods including, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

VI. Detection Methodologies

A. Detection of RNA

mRNA expression may be measured by any suitable method, including butnot limited to, those disclosed below.

In some embodiments, RNA is detection by Northern blot analysis.Northern blot analysis involves the separation of RNA and hybridizationof a complementary labeled probe. In other embodiments, RNA expressionis detected by enzymatic cleavage of specific structures (INVADER assay,Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543;6,001,567; 5,985,557; and 5,994,069; each of which is hereinincorporated by reference). The INVADER assay detects specific nucleicacid (e.g., RNA) sequences by using structure-specific enzymes to cleavea complex formed by the hybridization of overlapping oligonucleotideprobes. In still further embodiments, RNA (or corresponding cDNA) isdetected by hybridization to a oligonucleotide probe. A variety ofhybridization assays using a variety of technologies for hybridizationand detection are available. For example, in some embodiments, TaqManassay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos.5,962,233 and 5,538,848, each of which is herein incorporated byreference) is utilized. The assay is performed during a PCR reaction.The TaqMan assay exploits the 5′-3′ exonuclease activity of the AMPLITAQGOLD DNA polymerase. A probe consisting of an oligonucleotide with a5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye isincluded in the PCR reaction. During PCR, if the probe is bound to itstarget, the 5′-3′ nucleolytic activity of the AMPLITAQ GOLD polymerasecleaves the probe between the reporter and the quencher dye. Theseparation of the reporter dye from the quencher dye results in anincrease of fluorescence. The signal accumulates with each cycle of PCRand can be monitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

B. Detection of Protein

In other embodiments, protein may be detected by any suitable method. Insome embodiments, proteins are detected by immunohistochemistry. Inother embodiments, proteins are detected by their binding to an antibodyraised against the protein.

Antibody binding may be detected by many different techniques including,but not limited to, (e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotope labels,for example), Western blots, precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. In one embodiment, antibodybinding is detected by detecting a label on the primary antibody. Inanother embodiment, the primary antibody is detected by detectingbinding of a secondary antibody or reagent to the primary antibody. In afurther embodiment, the secondary antibody is labeled.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to cancer markers isutilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480; each of which is herein incorporated byreference.

C. Remote Detection Systems

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into datauseful for intracellular component contact mapping.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, wherein the information is provided toappropriate personnel. For example, in some embodiments of the presentinvention, a sample (e.g., a biopsy or a serum or urine sample) isobtained from a subject and submitted to an intracellular componentprofiling service (e.g., clinical lab at a medical facility, genomicprofiling business, etc.), located in any part of the world (e.g., in acountry different than the country where the subject resides or wherethe information is ultimately used) to generate raw data. Where thesample comprises a tissue or other biological sample, the subject mayvisit a medical center to have the sample obtained and sent to theprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

D. Detection Kits

In other embodiments, the present invention provides kits for thedetection and characterization of intracellular contact maps. In someembodiments, the kits contain antibodies specific for protein pairs inaddition to detection reagents (i.e., for example, SNAPPLE probes) andbuffers. In other embodiments, the kits contain reagents specific forthe detection of mRNA or cDNA (e.g., oligonucleotide probes or primers).In preferred embodiments, the kits contain all of the componentsnecessary to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results.

VII. Kits

In another embodiment, the present invention contemplates kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing elements according the describedmethods of this invention. The kit can optionally include a firstcontainer comprising a plurality binding partners, wherein each partneris attached to a different forked adapter molecule. The kit canoptionally include a second container comprising a solution capable offixing a biological cell sample. The kit can optionally include a thirdcontainer comprising buffers and reagents capable of supporting bindingof said binding partner to intracellular components of said fixedbiological cell sample. The kit can optionally include enzymes capableof performing PCR (i.e., for example, DNA polymerase, Tag polymeraseand/or restriction enzymes). The kit can optionally include apharmaceutically acceptable excipient and/or a delivery vehicle (e.g., aliposome). The reagents may be provided suspended in the excipientand/or delivery vehicle or may be provided as a separate component whichcan be later combined with the excipient and/or delivery vehicle. Thekits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe reagents by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the reagents inhow to identify said intracellular components bound to said bindingpartners. In particular, the instructions may describe construction ofan intracellular component contact map. While the instructionalmaterials typically comprise written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. Such media may include addresses to internetsites that provide such instructional materials.

EXPERIMENTAL Example I Conjugation of Antibodies to Nucleotides

Antibodies may be coupled to oligonucleotides using aldehyde/hydrazinechemistry (Solulink inc.), and purified by size exclusionchromatography, and then stored at +4° C. in PBSE with sodium azide.Kozlov, I. A. “Efficient strategies for the conjugation ofoligonucleotides to antibodies enabling highly sensitive proteindetection” Biopolymers 73:621-30 (2004). In general, affinity purifiedpolyclonal antibodies and antigen standards may be obtained from R&Dsystems and BD Biosciences. Sequences may be designed to minimizeprobe-probe heteroduplexes using mFold². A 1 mg batch of a polyclonalproximity probe may be sufficient for over 10 million analyses and thereagents are stable in storage.

Specifically, a pure antibody may be used without carrier proteins asBSA or gelatin. Dialyze the antibodies against PBS if they containazide. Concentrate by spin columns (Microcon YM-30, Amicon Cat no42410), if needed, to a concentration higher than 2 mg/ml.

1. Exchange buffer in the antibody to 55 mM Phosphate buffer, 150 mMNaCl, 20 mM EDTA, pH 7.2.

2. To 20 μg Antibody (10 μl) add 1 μl 4 mM sulfo-SMCC (diluted in DMSO)and incubate at room temp for 2 hrs.

3. Reduce 3 μl of 100 μM Oligo with 4 μl of 100 mM DTT in 50 μl of 55 mMPhosphate buffer, 150 mM NaCl 20 mM EDTA pH 7.2 for 1 hr at 37° C.

4. Equilibrate G-50 spin columns (Amersham Cat no 27-5330-02) with 55 mMPhosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.2

5. Do buffer exchange of both the sulfo-SMCC activated antibodies andreduced oligos with the G-50 columns from above. Repeat twice persample.

6. Mix the antibodies and oligos and dialyze (Slide-A-Lyzer, Pierce Catno 69562) against PBS, 5 mM EDTA pH 7.2 over night.

7. Collect the dialyzed conjugates and store them at +4° C.

Example II Plasma Samples

EDTA plasma samples may be collected and fresh frozen in aliquots at−80° C. Prior to analysis, PEG-8000 may be added to a finalconcentration of 5% and incubated at +4° C. for 30 min then centrifugedat 4,000 rpm for 20 minutes to remove potential assay interferences.

Example III Probe-Target Binding Incubation

One μL of each sample may be added to 1 μL of a probe mix resulting in a100 pM concentration of each probe in PBS pH 7.2, 20 μg/mL shearedpoly-A (Sigma), 2 mM EDTA, 1% BSA, 0.05% bulk goat IgG. Incubations wereperformed at 37° C. for 2 hours.

Example IV Ligation

120 μL ligation mixes may be added containing 100 nM connectingoligonucleotides, 2.5 units of Ampligase (Epicentre), 0.3 mM NADH⁺(Sigma), 10 mM DTT, 20 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂.Ligation proceeded at 30° C. for 15 minutes. Ligation was terminated byadding 0.25 μL of the uracil excision mix (Epicentre) degrading theuracil containing connectors.

Example V Amplification of a Barcoded Amplicon Pool

25 μL of the ligation reaction may be amplified in a 50-μL PCR using 200nM universal primers amplifying all sequences for 13 cycles. The productmay then be diluted 50-fold in 1×TE-buffer prior to real-time PCR.

Example VI Real-Time PCR

2 μLs of the diluted pre-amplification reaction may be added to the qPCRmixture to a volume of 10 μL containing iTaq qPCR Sybr Green master mixwith ROX (Bio-Rad) with 0.4 μM of respective target specific primer.Samples were run on a 384 well ABI 7900 with the default cyclingprotocol.

We claim:
 1. A composition comprising a forked adapter moleculecomprising a first end and a second end, wherein said first endcomprises two single stranded portions and said second end is doublestranded, wherein one of said two single stranded portions is attachedto a single binding partner, wherein said second end comprises acomponent-specific oligonucleotide barcode duplex sequence wherein saidbarcode sequence comprises a nucleic acid sequence unique to saidcomponent-specific binding partner, wherein said molecule does not havea fluorescent reporter.
 2. The composition of claim 1, wherein saidbinding partner is selected from the group consisting of an antibody, alocked nucleic acid, a receptor, and a small organic molecule.
 3. Thecomposition of claim 1, wherein said first end further comprises alinker molecule.
 4. The composition of claim 3, wherein said linkermolecule attaches said first end to said binding partner.
 5. Acomposition comprising a nucleic acid sequence, wherein said nucleicacid sequence comprises a 3′-5′ first strand and a 5′-3′ second strand,wherein said first strand and said second strand join a firstintracellular component-specific oligonucleotide barcode and a secondintracellular component-specific oligonucleotide barcode thereby formingan asymmetric barcode region wherein said first strand 3′ end isattached to a single first binding partner having specific affinity forsaid first intracellular component and said second strand 3′ end isattached to a single second binding partner having specific affinity forsaid second intracellular component, wherein said asymmetric barcoderegion comprises a nucleic acid sequence unique to said first and secondintracellular components.
 6. The composition of claim 5, wherein saidfirst strand 3′ end further comprises a first sequence complementary toa first primer.
 7. The composition of claim 5, wherein said first strand5′ end further comprises a second sequence complementary to a secondprimer.
 8. The composition of claim 5, wherein said first bindingpartner is selected from the group consisting of an antibody, a lockednucleic acid, a receptor, and a small organic molecule.
 9. Thecomposition of claim 5, wherein said second binding partner is selectedfrom the group consisting of an antibody, a locked nucleic acid, areceptor, and a small organic molecule.
 10. A kit, comprising: a) afirst container comprising a plurality of forked adapter moleculescomprising a first end and a second end, wherein said first endcomprises two single stranded portions and said second end is doublestranded, wherein one of said two single stranded portions is attachedto a single binding partner, wherein said second end comprises acomponent-specific oligonucleotide barcode duplex sequence wherein saidbarcode sequence comprises a nucleic acid sequence unique to saidcomponent-specific binding partner, wherein said molecule does not havea fluorescent reporter. b) a second container comprising a solutioncapable of fixing a biological cell sample; c) a third containercomprising buffers and reagents capable of supporting binding of saidbinding partner to intracellular components of said fixed biologicalcell sample; and d) a set of instructions describing: i) how to identifysaid intracellular components bound to said binding partners; and ii)construction of an intracellular component contact map.
 11. The kit ofclaim 10, wherein said binding partners are selected from the groupconsisting of antibodies, locked nucleic acids, intracellular receptors,and small organic molecules.
 12. A composition consisting of a firstnucleic acid sequence comprising a first duplex region encoding a firstbarcode sequence unique to an attached first intracellular componentbinding partner, and a second nucleic acid sequence comprising a secondduplex region encoding a second barcode sequence unique to an attachedsecond intracellular component binding partner, and wherein said firstand second binding partners have specific affinity for differentintracellular components.
 13. The composition of claim 12, wherein saidfirst nucleic acid sequence or said second nucleic acid sequencecomprise a phosphorylated blunt end.
 14. The composition of claim 12,wherein said first nucleic acid sequence and said second nucleic acidsequence comprise a self-complementary sequence.
 15. The composition ofclaim 12, wherein said first nucleic acid sequence comprises a firstprimer.
 16. The composition of claim 15, wherein said first primer is afirst sequencing primer.
 17. The composition of claim 12, wherein saidfirst nucleic acid sequence comprises a nucleic acid sequencecomplementary to a second primer.
 18. The composition of claim 17,wherein said second primer is a second sequencing primer.
 19. Thecomposition of claim 12, wherein said first binding partner is selectedfrom the group consisting of an antibody, a locked nucleic acid, areceptor, and a small organic molecule.
 20. The composition of claim 12,wherein said second binding partner is selected from the groupconsisting of an antibody, a locked nucleic acid, a receptor, and asmall organic molecule.
 21. The composition of claim 12, wherein saidligated first and second nucleic acids comprise an asymmetric barcoderegion.
 22. The composition of claim 21, wherein said asymmetric barcoderegion unambiguously identifies said different intracellular components.23. A forked adapter molecule consisting of a first end and a secondend, wherein said first end has two single stranded portions and saidsecond end is double stranded, wherein one of said two single strandedportions is attached to a single binding partner, said binding partnerhaving a specific affinity for a single intracellular component, whereinsaid second end is attached to an intracellular component-specificoligonucleotide barcode duplex sequence wherein said barcode sequence isa nucleic acid sequence unique to said intracellular component.
 24. Anucleic acid sequence consisting of a 3′-5′ first strand and a 5′-3′second strand, wherein said first strand and said second strand join afirst intracellular component-specific oligonucleotide barcode and asecond intracellular component-specific oligonucleotide barcode therebyforming an asymmetric barcode duplex region wherein said first strand 3′end is attached to a single first binding partner having specificaffinity for said first intracellular component and said second strand3′ end is attached to a single second binding partner having specificaffinity for said second intracellular component, wherein saidasymmetric barcode duplex region is a nucleic acid sequence unique tosaid first and second intracellular components.